Metabolomics signatures of a subset of RET variants according to their oncogenic risk level

in Endocrine-Related Cancer
Correspondence should be addressed to C Veyrat-Durebex: charlotte.veyrat@live.fr
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Thirty percent of medullary thyroid carcinomas (MTCs) are related to dominant germline pathogenic variants in the RET proto-oncogene. According to their aggressiveness, these pathogenic variants are classified in three risk levels: ‘moderate’, ‘high’ and ‘highest’. The present study compares the metabolomics profiles of five pathogenic variants, whether already classified or not. We have generated six stable murine fibroblast cell lines (NIH3T3) expressing the WT allele or variants of the human RET gene, with different levels of pathogenicity, including the M918V variant that is yet to be accurately classified. We carried out a targeted metabolomics study of the cell extracts with a QTRAP mass spectrometer, using the Biocrates Absolute IDQ p180 kit, which allows the quantification of 188 endogenous molecules. The data were then subjected to multivariate statistical analysis. One hundred seventy three metabolites were accurately measured. The metabolic profiles of the cells expressing the RET variants were found to be correlated with their oncogenic risk. In addition, the statistical model we constructed for predicting the oncogenic risk attributed a moderate risk to the M918V variant. Our results indicate that metabolomics may be useful for characterizing the pathogenicity of the RET gene variants and their levels of aggressiveness.

Downloadable materials

  • Supplementary figure S1 Western blot of RET protein in cell lines expressing mutant or wild-type RET. The #x03B1;-tubulin protein was used as a loading control and the ratio of RET/#x03B1;-tubulin was calculated for each cell line.
  • Oxygen Consumption Rate (pmol/min/2.104 cells)
  • List of metabolites detected in samples using Biocrates kit

 

      Society for Endocrinology

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    (A) Cell morphology. Pictures of the cell cultures stably transfected with RET WT or mutant (C634R, L790F, V804M, M918V and M918T). Scale bars represent 300 µm. (B) Cell signaling. Western blots of ERK, p-ERK, AKT and p-AKT proteins in cell lines expressing mutant or WT RET and the ratio of phosphorylated protein related to non-phosphorylated were calculated. (C) Cell growth. The cell growth (percentage compared to RET-WT NIH3T3 cells considered as 100%) of cell cultures stably transfected with RET mutants (C634R, L790F, V804M, M918V and M918T), after 72 h of GDNF treatment. All RET mutant cells had a significantly higher growth rate compared to WT cells (*P < 0.01); and RET-M918T cells had a higher growth rate compared to all the other mutant cells (¥ P = 0.0002).

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    Cells expressing human RET have distinct metabolomics signatures. (A) PCA plot obtained from metabolomics profiles of all the samples studied: non-transfected NIH3T3 cells (NIH3T3-NT) (grey dots), transfected with RET-WT (black dots), or mutant RET (C634R green dots, L790F light blue dots, V804M blue dots, M918T red dots and M918V light red dots). R 2 X = 0.841 and Q 2 = 0.796. (B) OPLS-DA plot of the model performed according to the seven cell types (non-transfected or transfected with RET plasmids). Non-transfected NIH3T3 cells (NIH3T3-NT) (grey dots); cells transfected with RET-WT (black dots), or mutant RET (C634R green dots, L790F light blue dots, V804M blue dots, M918T red dots and M918V light red dots). R 2 X = 0.89, R 2 Y = 0.849 and Q 2 = 0.616. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0314.

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    Cells expressing mutant or WT RET have distinct metabolic profiles. (A) The OPLS-DA plot based on cells overexpressed mutant or WT RET. NIH3T3 cells transfected with WT RET (blue dots) or with mutant RET (green dots). R 2 X = 0.819, R 2 Y = 0.963 and Q 2 = 0.839. (B) Volcano plot of the univariate analysis of the metabolomics data of the two groups (WT and mutant RET), using fold change >2 and P-value <0.05 as thresholds. Graph was built with between-group metabolite fold change values on the x-axis, and log-scaled P-values on the y-axis. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0314.

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    The metabolomics profiles of cells expressing mutated human RET differ according to the risk level. (A) Score plot of the OPLS-DA model performed according to the level of oncogenic risk of RET mutations and obtained from the metabolomics profiles of NIH3T3 cells transfected with various mutants of RET: C634R as a ‘high’ risk mutant (green dots), L790F and V804M as ‘moderate’ risk mutants (blue dots), and M918T as the ‘highest risk’ mutant (red dots). R 2 X = 0.879, R 2 Y = 0.958, and Q 2 = 0.87. (B) Metabolic pathway analysis of discriminant metabolites according to the risk level of RET mutations. Metabolic pathway analyses related to the metabolites that were discriminant in the OPLS-DA of the level of oncogenic risk of RET mutations in cell lines using the MetaboAnalyst tools. The figure graphically summarizes the analysis of metabolite set enrichment for discriminant metabolites. The horizontal bars summarize the main metabolite sets identified in this analysis; the bars are colored according to their P-values, and the length is based on the fold enrichment. A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0314.

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    Prediction of the risk level of NIH3T3 RET-M918V cells. Score plot of the prediction of the risk level of NIH3T3 RET-M918V cells using the OPLS-DA model for the level of oncogenic risk of RET mutations obtained from the metabolomics profiles of NIH3T3 cells transfected with various mutants of RET (Fig. 4A): C634R as a ‘high’ risk mutant (green dots), L790F and V804M as ‘moderate’ risk mutants (blue dots), M918T as the ‘highest risk’ mutant (red dots), and M918V as unclassified mutant (light orange dots). 90% of RET-M918V cells are predicted to be at moderate risk (black circle). A full color version of this figure is available at https://doi.org/10.1530/ERC-18-0314.

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