MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state

Abstract

Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.

Keywords: N-terminal processing; cotranslational modifications; glutathione redox homeostasis; methionine aminopeptidase; quantitative targeted proteomics.

Figure 1. Selective cytostatic effect of MetAP2 inhibition

A. Analysis of cell proliferation in the presence of various concentrations of fumagillin, for eleven mammalian cell lines. Cells were cultured in presence of the drug for 72 h and their proliferation was then assessed. Cell lines are classified according to their sensitivity to fumagillin. HUVEC, U87, U937, HaCaT and A549 cells were sensitive to fumagillin. MDA-MB-231 and THP-1 cells were considered to be moderately sensitive to fumagillin. K562, H1299, Jurkat and HCT116 cells were insensitive to fumagillin. B. Cell-cycle analysis of HUVEC treated with 5 nM fumagillin for 24, 48 or 72 h (Fum, black bars) or mock-treated (CT, white bars). Cell accumulation at the various steps in the cell cycle is shown. Error bars represent the standard error of the mean, n=3-5, p-Value from two-sided t-test are represented as described in Methods section. Fum, fumagillin.

Figure 2. MetAP2 inhibition induces iMet retention of inefficient MetAP substrates

Large-scale mass spectrometry analysis of NME efficiency with various cell lines. A. Overall NME efficiency is shown for the HUVEC, U87, K562 and HCT116 lines. For each experiment, Uniprot protein accessions were run through the NME predictor (TermiNator, [55]) for comparison. The results are classified by control (CT) and fumagillin-treated samples (Fum). B. For each cell line, N-terminal peptides were clustered on the basis of their second amino acids, as represented by the one-letter code, on the x axis. For each class, NME efficiency was calculated as the percentage of N-terminal peptides with a cleaved iMet. Data are plotted as a percentage of the total number of peptides identified for the class concerned. Each graph includes results for a control sample (white bars), a fumagillin-treated sample (gray bars) and the results of TermiNator prediction based on the data for control samples. For each line a close up view is given for V and T classes for clarity.

Figure 3. M[T/V]X substrates require the full set of active MetAPs for iMet cleavage

For each cell line and set of treatment conditions, the efficiency of NME for proteins with specific N-terminal sequences is analyzed further. The proportion of proteins without the iMet is shown in black, the white bar shows the proportion of proteins with a retained iMet and the gray bar represents proteins with partial cleavage of the iMet residue. A. MGX and MAPX proteins, B. MPX, MSX and MMX classes and C. MTX and MVX classes.

Figure 4. MetAP1 and MetAP2 quantification: both proteins are more abundant in the insensitive cell line K562

A. Relative quantification of MetAP1 and MetAP2. The ratios of the summed areas of all transitions of the light over the heavy-labelled peptide (L/H) are shown for sample-preparation quadruplicates for the 6 targeted peptides in three different cell types. B. Calibration curve for the absolute quantification of MetAP2 by monitoring the IDFGTHISGR peptide. The calibration curve was done using the points where CVs were lower than 15% and the accuracy was between 80-120% (full diamonds) the lowest point being the limit of quantification (LOQ). The points not meeting these criteria were discarded (empty diamonds). The MetAP2 protein is present in a very low abundance in all three cell lines K562 (box), HUVEC (triangle, below LOQ) and U87 (disk, below LOQ). Both independent studies showed the same trend: MetAP2 is more abundant in fumagillin-insensitive cell lines. The statistical significance of the differences in MetAP2 quantity is given by a two-sample t-test between each line. The p-Value is given as described in the Methods section.

Figure 5. Cell-specific accumulation of MetAP mRNAs correlates with fumagillin sensitivity

The eleven cell lines were cultured according the standard procedure. Total RNAs were extracted and the MetAP2 A. and MetAP1 B. mRNAs were quantified. MetAP mRNAs were quantified with two reference genes from the ten tested. Results are normalized with respect to data for the U87 cell line and are expressed as a fold-change. The data presented are the means from at least three independent experiments. Half maximal effective concentration (EC₅₀) values were obtained from the dose-response curves shown in Figure 1A. Error bars represent the standard error of the mean of 3 to 4 experiments. The statistical significance (two-sided t-test) of the differences when comparing U87 (*) and HUVEC (#) lines to the others is displayed. p-Value is shown as follow: * or # pVal<005, ** or ## pVal<0.01, *** pVal<0

Figure 6. MetAP2 inhibition selectively alters glutathione redox state in fumagillin sensitive cells

A. HUVEC and HCT116 cells were cultured without (CT) or with 5 nM and 1 μM fumagillin (Fum) for respectively 72 h before glutathione quantification. The amount shown for glutathione corresponds to the total pool of glutathione (top panels); GSSG is the oxidized form of glutathione (middle panels) and the glutathione ratio is the ratio of reduced to oxidized glutathione (bottom panels). Error bars represent the standard error of the mean. B. Cell cycle analysis for HUVECs left untreated (CT) or treated (Fum) with 5 nM fumagillin for 24, 48 or 72 h or with fumagillin and N-acetylcysteine (NAC) for 72 h. Cell accumulation at the various steps of the cell cycle is shown. Error bars represent the standard error of the mean. p-Value from two-sided t-test are represented as described in Methods section.

Figure 7. Understanding the cell selectivity of MetAP2 inhibition phenotype

The y-axis shows total cellular MetAP activity. In control conditions (CT), MetAP1 and MetAP2 are active and catalyze the complete cleavage of MetAP substrates. Upon MetAP2 inhibition, only MetAP1 can process MetAP substrates. Due to their very similar substrate specificities, MetAP1 can cleave most of the MetAP2 substrates. Thus, provided that MetAP1 is not limiting, substrates are processed (NS1). However, if MetAP1 becomes limiting within the cell, then either the unprocessed substrates are not important for cell growth (NS2) or some specific substrates may become less stable, with effects on growth (S1). In such conditions, the first proteins affected are the poorest MetAP substrates (M[V/T]; NS2 + S1). These proteins may be involved in glutathione redox homeostasis and control important aspects of cell life. If smaller amounts of MetAP1 are present in the cell, then a larger number of substrates are likely to be affected, including abundant proteins beginning with MG (S2). Regardless of cell sensitivity, MetAP2 inhibition impairs glutathione redox homeostasis. This defect is overcome or compensated in insensitive lines but not in sensitive lines, resulting in a slow-growth phenotype. If MetAP activity decreases further, too many substrates are likely to be affected, with deleterious effects on cell function, eventually leading to cell death (D).