Understanding The Link Between Glutamine Metabolism And Angiogenesis In Astrocytoma

Angiogenic switch is a hallmark for tumor growth, and strategies to inhibit this process have been pursued for cancer therapy, but with limited long-term efficacy. Recently, targeting endothelial cell (EC) metabolism emerged as an alternative to regulate angiogenesis. In fact, glutamine metabolism is essential for EC sprouting in vitro, and the consumption/secretion rate measurements revealed that proliferating ECs consumed glutamine more than any other amino acid. Glutamine is transported into the cells and converted into α-ketoglutarate to enter the tricarboxylic acid cycle through glutaminolysis, which is upregulated in cancer. Glutaminase (coded by GLS), the main regulator of this pathway, presents two isoforms: GLSiso1 and GLSiso2. We have analyzed the expression level of both GLS isoforms in different grades of astrocytoma, with a gradual increase of their expressions in parallel to the malignancy. GLSiso1 was highly expressed in normal brain samples, in contrast to low GLSiso2 expression, indicating GLSiso2 as a more eligible therapeutic target. GLSiso2-specific transcript inhibition by siRNA in GBM U87MG cells lead to significant decrease of tumor cell proliferation, and a temozolomide-sensitizing effect, relative to NTC-controls. The RNASeq transcriptome analysis of these GLSiso2-silenced cells revealed a differential expression of genes related to blood vessel development, extracellular matrix organization, angiogenesis and tube development with FDR of 4.95e-04, 5.25e-06, 5.92e-05, 8.6e-06 and 1.0e-04, respectively. The immunohistochemistry analysis in human astrocytoma FFPE samples demonstrated GLS positivity on tumor cells and blood vessels, with higher protein expression in more malignant astrocytoma. Such observation corroborated the transcriptomic finding of GLSiso2 participation in the EC compartment. Moreover, the system biology analysis revealed an interesting net among GLS, TP53, ITB1, TGFBI, TGFBR1/2, COL1A1/2, COL5A2, LUM, ENG, ANGPT1 connecting genes related to metabolism, extracellular matrix and angiogenesis. In the search for the most suitable EC line to study the impact of GLS isoforms on the vessel compartment, we observed that HUVEC immortalized umbilical EC line presented higher expression of GLSiso2 than GLSiso1. Chemical blockade of the total GLS by BPTES and 968 was tested in HUVEC cells and a significant proliferation and migration decrease were observed. Further GLSiso2 specific silencing of ECs might clarify the crosstalk between tumor cells and ECs, and will allow the identification of key targets in their metabolic reprogramming during tumor growth. Citation Format: Yollanda E. Franco, Roseli S. Soares, Marina T. Lima, Antonio M. Lerario, Sueli M. Shinjo, Suely K. Marie. Understanding the link between glutamine metabolism and angiogenesis in astrocytoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3739.