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NMR based metabolomics study of Y2 receptor activation by neuropeptide Y in the SK-N-BE2 human neuroblastoma cell line

ABSTRACT: Neuropeptide Y (NPY) and its Y2 receptors (Y2R) play an important role in regulating growth of various tumors and are highly expressed in human neuroblastoma brain tumors. Cell lines derived from neuroblastoma brain tumors provide a good model to study the consequences of NPY activation of Y2R in tumor metabolism. In this study, the metabolic response to activation and inhibition of Y2R was examined in the human neuroblastoma cell line SK-N-BE2 using high-resolution nuclear magnetic resonance spectroscopy (NMR). Three treatments were evaluated: (1) activation of Y2R with NPY, (2) blocking Y2R with BIIE0246, and (3) treatment of cells with both NPY and BIIE. The largest metabolic changes were observed for NPY activation of Y2R. The principal response to NPY activation of Y2R was increased glycolysis (Warburg effect). Our results also showed depleted intracellular nutrients in NPY activated cells, which may have been due to the high rate of conversion of glucose to lactate, glycine and alanine. All amino acids except glutamine were increased in response to Y2R activation, implying increased autophagic degradation of proteins. TCA cycle intermediates were not detected, possibly due to low steady-state concentrations, but the increase of glutamate and its high correlation with glucose provided evidence of increased TCA activity. The changes of most metabolites observed upon NPY treatment were reversed with BIIE treatment. In summary, our study indicates that NPY activation of Y2R in human neuroblastoma cells stimulates glycolysis, glutaminolysis and possibly TCA activity.

INSTRUMENT(S): Bruker

SUBMITTER: Bo Wang

PROVIDER: MTBLS104 | MetaboLights | 2014-10-01

REPOSITORIES: MetaboLights

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Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

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NMR based metabolomics study of Y2 receptor activation by neuropeptide Y in the SK-N-BE2 human neuroblastoma cell line

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