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. 2017 Aug 29:5:7.
doi: 10.1186/s40170-017-0169-9. eCollection 2017.

"VSports在线直播" Bortezomib resistance in multiple myeloma is associated with increased serine synthesis

Esther A Zaal  1 , Wei Wu  1 , Gerrit Jansen  2 , Sonja Zweegman  3 , Jacqueline Cloos  3   4 , Celia R Berkers  1
Affiliations

Bortezomib resistance in multiple myeloma is associated with increased serine synthesis

Esther A Zaal et al. Cancer Metab. .

Abstract

Background: The proteasome inhibitor bortezomib (BTZ) is successfully applied in the treatment of multiple myeloma, but its efficacy is restricted by the wide-spread occurrence of resistance VSports手机版. Metabolic alterations play an important role in cancer development and aid in the cellular adaptation to pharmacologically changed environments. Metabolic changes could therefore play an essential role in the development of drug resistance. However, specific metabolic pathways that can be targeted to improve bortezomib therapy remain unidentified. .

Methods: We elucidated the metabolic mechanisms underlying bortezomib resistance by using mass spectrometry-based metabolomics and proteomics on BTZ-sensitive and BTZ-resistant multiple myeloma cell lines as well as in a set of CD138+ cells obtained from multiple myeloma patients V体育安卓版. .

Results: Our findings demonstrate that a rewired glucose metabolism sustains bortezomib resistance V体育ios版. Mechanistically, this results in higher activity of both the pentose phosphate pathway and serine synthesis pathway, ultimately leading to an increased anti-oxidant capacity of BTZ-resistant cells. Moreover, our results link both serine synthesis pathway activity and expression of 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of serine synthesis, to bortezomib resistance across different BTZ-resistant multiple myeloma cell lines. Consistently, serine starvation enhanced the cytotoxicity of bortezomib, underscoring the importance of serine metabolism in the response to BTZ. Importantly, in CD138+ cells of clinically bortezomib refractory multiple myeloma patients, PHGDH expression was also markedly increased. .

Conclusions: Our findings indicate that interfering with serine metabolism may be a novel strategy to improve bortezomib therapy and identify PHGDH as a potential biomarker for BTZ resistance. VSports最新版本.

Keywords: Bortezomib; Drug resistance; Metabolism; Multiple myeloma; PHGDH V体育平台登录. .

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Conflict of interest statement

Ethics approval and consent to participate

Research was approved by the Medical Ethics Committee of the VU University Medical Center and all patients gave written informed consent.

Consent for publication

Not applicable

Competing interests

All authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

"VSports最新版本" Figures

Fig. 1
Fig. 1
Bortezomib resistance is not solely driven by adaptations of the proteasome. a Cell viability of RPMI-8226 wild type (WT) and bortezomib-resistant (BTZ/7 and BTZ/100) cells after a 48-h treatment with increasing concentrations of bortezomib. Results represent % cell viability ± SD compared to non-treated controls of a representative experiment (n = 3). b In gel fluorescence measurements of a representative experiment showing proteasome activity profiles of RPMI-8226 WT, BTZ/7, and BTZ/100 cells after a 1-h incubation with proteasome activity probe Me4BodipyFL-Ahx3L3VS (lower panel). Quantification of gel images, with subunit activity plotted as of total proteasome activity. Results represent averages of 3 independent experiments (upper panel). c Total proteasome activity of RPMI-8226 WT, BTZ/7 and BTZ/100 cells after a 2-h incubation with increasing concentrations of bortezomib, compared to non-treated controls. Results represent quantification of gel images obtained by a 1-h incubation with Me4BodipyFL-Ahx3L3VS. Results represent averages of 3 independent experiments. BTZ = bortezomib
Fig. 2
Fig. 2
Bortezomib-resistant cells have an enhanced activity of the pentose phosphate pathway. Intra- and extracellular metabolite analysis of RPMI-8226 wild type (WT) and bortezomib resistant (BTZ/100) cells. Cells were suspended in DMEM containing 8 mM [U-13C] D-glucose. a, b Media samples were collected after 8 h, followed by LC-MS analysis of extracellular glucose (a) and lactate (b). Results represent % peak area ± SD compared to cell-free media (n = 3). Unpaired t-tests were performed (* = p < 0.05, ** = p < 0.01,). c Intracellular metabolites were extracted after 4 and 8 h and analyzed by LC-MS. Data are means ± SD (n = 3) of unlabeled (white) and 13C- labeled metabolites (gray). AMP = adenosine monophosphate, GMP = guanosine monophosphate, PPP = pentose phosphate pathway, TCA = tricarboxylic acid
Fig. 3
Fig. 3
Enhanced pentose phosphate pathway activity increases the anti-oxidant capacity of bortezomib-resistant cells. a Total GSH levels of RPMI-8226 wild type (WT) and bortezomib resistant (BTZ/100) cells. Data are means ± SD (n = 3) b Cell viability of RPMI-8226 WT and BTZ/100 cells after a 16-h treatment with increasing concentrations of H2O2. Results represent mean cell viability ± SD compared to non-treated controls (n = 3). c Cell viability of RPMI-8226 BTZ/100 cells after treatment for 24 h with 50 μM trans-androsterone (TA), followed by 24 h with increasing concentrations of H2O2. Results represent mean cell viability ± SD compared to non-treated control (n = 3). d GSH/GSSG ratio of RPMI-8226 WT and BTZ/100 cells after 5 min treatment with 5 mM DHA. Data are means ± SD (n = 3). e Analysis of intracellular levels of DHA (left panel) and AA (right panel) in RPMI-8226 WT and BTZ/100 cells. Cells were treated with 5 mM DHA or vehicle for 5 min and subjected to LC-MS analyses. Data are means ± SD (n = 3). Two-way ANOVA tests were performed (ns = not significant, * = p < 0.05). AA = ascorbic acid, DHA = dehydroascorbic acid, GSH = glutathione, TA = trans-androsterone
Fig. 4
Fig. 4
Bortezomib resistant cells have a higher activity of the serine synthesis pathway. a, b Intra- and extracellular analysis of serine levels of RPMI-8226 wild type (WT) and bortezomib resistant (BTZ/100) cells. Cells were suspended in DMEM containing 8 mM [U-13C] D-glucose. Intracellular metabolites were extracted after 4, 8 and 24 h and analyzed by LC-MS (a). Data are means ± SD (n = 3) of unlabeled (white) and 13C- labeled serine (left panel). Labeled serine was plotted as M+1, M+2 and M+3 isotopomers (right panel). Media samples were collected after 8 and 24 h, followed by LC-MS analysis of extracellular serine (b). Results represent % peak area ± SD compared to non-treated media (n = 3). c Intracellular metabolite analysis of RPMI-8226 WT and BTZ/100 cells in the presence or absence of extracellular serine and glycine (−SG). Cells were grown complete medium in the presence or absence of 0.4 mM serine. After 24 h, media were replaced with matched media containing 20 mM [U-13C] D-glucose and intracellular metabolites were extracted after 4 h, followed by LC-MS analysis. Data are means ± SD (n = 3) of labeled metabolites. d Cell viability of RPMI-8226 WT and BTZ/100 cells after 48 h of serine starvation (−SG). Results represent mean cell viability ± SD compared to non-treated control (n = 3). e Cell viability of RPMI-8226 WT cells after 48 h in 0.4 mM, 0.1 mM or no serine, including 24 h of 10 nM bortezomib. Results represent mean cell viability ± SD compared to non-treated control (n = 3). One-way ANOVA tests were performed (ns = not significant, * = p < 0.05, ** = p < 0.01, **** = p < 0.0001). BTZ = bortezomib, GSH = glutathione
Fig. 5
Fig. 5
Bortezomib resistance correlates to the expression of PHGDH. a Graphical representation of quantitative proteomics data. Proteins are ranked in volcano plot according to their statistically p-value (y-axis) and relative abundance ratio between RPMI-8226 wild type (WT) and bortezomib resistant (BTZ/100 cells) (x-axis). Colored spots represent significantly upregulated (red) or downregulated (green) proteins in BTZ/100 cells with at least a 5-fold change. Significantly regulated metabolic enzymes are marked. b Quantitative proteomics data of enzymes involved in the serine synthesis pathway. Data represents means ± SD (n = 3). c Immunoblot of 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH) and Tubulin expression in RPMI-8226, AMO-1 and ARH-77 bortezomib-sensitive and -resistant cells. d Fractions of serine M+3 in RPMI-8226, AMO-1 and ARH-77 bortezomib-sensitive and -resistant cells. Data represent % serine M+3 of total serine ± SD (n = 3). One-way ANOVA tests were performed (**** = p < 0.0001). e Correlation between serine M+3 fractions and bortezomib sensitivity in RPMI-8226, AMO-1 and ARH-77 bortezomib-sensitive and -resistant cells. IC50s were determined in cell viability assay after 48 h of increasing concentrations of bortezomib. Pearson correlation r = 0.56 (p = .0042). f Immunoblot of PHGDH, PSAT1 and PSPH in isolated CD138+ plasma cells from diagnosed multiple myeloma patients (n = 6). Patients with progressive disease/refractory to BTZ-containing therapy are indicated with *. PHGDH is blotted on a separate membrane. BTZ = bortezomib, CFZ = carfilzomib, PHGDH = 3-phosphoglycerate dehydrogenase, PSAT1 = phosphoserine aminotransferase 1, PSPH = phosphoserine phosphatase, SSP = serine synthesis pathway
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