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

Grant number: 14/17820-3
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): January 01, 2015
Effective date (End): February 28, 2018
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal researcher:Sandra Martha Gomes Dias
Grantee:Douglas Adamoski Meira
Home Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovações (Brasil). Campinas , SP, Brazil
Associated research grant:09/10875-9 - Cellular and biochemical studies of the glutaminase enzyme and its relation with cancer, AP.JP


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)

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Scientific publications (6)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DIAS, MARILIA M.; ADAMOSKI, DOUGLAS; DOS REIS, LARISSA M.; ASCENCAO, CAROLLINE F. R.; DE OLIVEIRA, KRISHINA R. S.; PASCHOALINI MAFRA, ANA CAROLINA; DA SILVA BASTOS, ALLINY CRISTINY; QUINTERO, MELISSA; CASSAGO, CAROLINA DE G.; FERREIRA, IGOR M.; et al. GLS2 is protumorigenic in breast cancers. Oncogene, v. 39, n. 3, p. 690-702, . (13/05668-0, 14/18061-9, 12/14298-9, 13/23510-4, 14/17820-3, 14/06512-6, 14/15968-3, 15/25832-4, 12/09452-9, 14/20673-2, 16/06625-0, 12/11577-4, 11/10127-2)
QUINTERO, MELISSA; ADAMOSKI, DOUGLAS; DOS REIS, LARISSA MENEZES; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; SOUSA DE OLIVEIRA, KRISHINA RATNA; GONCALVES, KALIANDRA DE ALMEIDA; DIAS, MARILIA MEIRA; CARAZZOLLE, MARCELO FALSARELLA; GOMES DIAS, SANDRA MARTHA. Guanylate-binding protein-1 is a potential new therapeutic target for triple-negative breast cancer. BMC CANCER, v. 17, . (14/17820-3, 12/11577-4, 13/23510-4, 09/53853-5, 14/15968-3, 15/25832-4, 14/06512-6, 12/09452-9, 14/18061-9)
KRISHNA NAGAMPALLI, RAGHAVENDRA SASHI; NECIOSUP QUESNAY, JOSE EDWIN; ADAMOSKI, DOUGLAS; ISLAM, ZEYAUL; BIRCH, JAMES; SEBINELLI, HEITOR GOBBI; BRUNO MOREIRA GIRARD, RICHARD MARCEL; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; FALA, ANGELA MARIA; PAULETTI, BIANCA ALVES; et al. Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter. SCIENTIFIC REPORTS, v. 8, . (17/02391-8, 14/20673-2, 16/06034-2, 15/02734-7, 15/25832-4, 14/17820-3, 14/12663-7, 14/06954-9)
DOS REIS, LARISSA MENEZES; ADAMOSKI, DOUGLAS; OLIVEIRA SOUZA, RODOLPHO ORNITZ; RODRIGUES ASCENCAO, CAROLLINE FERNANDA; SOUSA DE OLIVEIRA, KRISHINA RATNA; CORREA-DA-SILVA, FELIPE; DE SA PATRONI, FABIO MALTA; DIAS, MARILIA MEIRA; CONSONNI, SILVIO ROBERTO; MENDES DE MORAES-VIEIRA, PEDRO MANOEL; et al. Dual inhibition of glutaminase and carnitine palmitoyltransferase decreases growth and migration of glutaminase inhibition-resistant triple-negative breast cancer cells. Journal of Biological Chemistry, v. 294, n. 24, p. 9342-9357, . (17/06225-5, 14/17820-3, 13/23510-4, 14/15968-3, 16/06034-2, 15/25832-4, 14/06512-6, 15/26059-7, 15/15626-8, 14/18061-9)
REDIS, ROXANA S.; VELA, LUZ E.; LU, WEIQIN; DE OLIVEIRA, JULIANA FERREIRA; IVAN, CRISTINA; RODRIGUEZ-AGUAYO, CRISTIAN; ADAMOSKI, DOUGLAS; PASCULLI, BARBARA; TAGUCHI, AYUMU; CHEN, YUNYUN; et al. Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2. MOLECULAR CELL, v. 61, n. 4, p. 520-534, . (14/17820-3, 14/15968-3, 14/20673-2)
COSTA, RENNA K. E.; RODRIGUES, CAMILA T.; CAMPOS, JEAN C. H.; PARADELA, LUCIANA S.; DIAS, MARILIA M.; DA SILVA, BIANCA NOVAES; DE VALEGA NEGRAO, CYRO VON ZUBEN; GONCALVES, KALIANDRA DE ALMEIDA; ASCENCAO, CAROLLINE F. R.; ADAMOSKI, DOUGLAS; et al. High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules. ACS PHARMACOLOGY & TRANSLATIONAL SCIENCE, v. 4, n. 6, p. 1849-1866, . (13/23510-4, 15/25832-4, 16/09077-4, 13/07600-3, 19/16351-3, 14/17820-3, 14/15968-3)
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
MEIRA, Douglas Adamoski. Post-transcriptional regulation of glutaminase enzyme by HuR and its relationship with high glutaminolytic levels in tumors. 2018. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.

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