Methylglyoxal, a glycolysis side-product, induces Hsp90 glycation and YAP-mediated tumor growth and metastasis
- University of Liège, Belgium
- Maastricht University, Netherlands
- Cardiovascular Research Institute Maastricht, The Netherlands
- University of Nagoya, Japan
- Yale University, United States
- University of Melbourne, Australia
- Association for Research and Treatments Against Cancer, France
Figures
Figure 1
High carbonyl stress and nuclear YAP are positively associated in human breast cancer.
(A) In cancer cells, a high glycolytic metabolism and/or a decrease of the MG-detoxifying activity of Glyoxalase 1 (Glo1) lead to high MG level thus establishing a carbonyl stress. (B) YAP IHC …
Figure 2 with 3 supplements
Methylglyoxal induces YAP persistence in confluent breast cancer cells.
(A) Immunofluorescence (IF) staining shows that YAP (Santa Cruz antibody, H125) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density …
Figure 2—figure supplement 1
Methylglyoxal induces YAP accumulation in confluent breast cancer cells.
(A and D) Immunofluorescence staining shows that YAP (Santa Cruz antibody, H125) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density …
Figure 2—figure supplement 2
Methylglyoxal induces YAP accumulation in confluent breast cancer cells.
(A, B and C) Immunofluorescence (IF) staining shows that YAP (Cell Signaling antibody, 4912) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high …
Figure 2—figure supplement 3
Methylglyoxal induces TAZ accumulation in confluent breast cancer cells.
(A, B, and C) Immunofluorescence (IF) staining shows that TAZ is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density (Confluent) in …
Figure 3 with 4 supplements
High endogenous MG induces YAP nuclear accumulation in breast cancer cells.
(A) Detection of MG was performed using MBo specific fluorescent probe, as described in Materials and Methods section, and showed MG cellular increase in MDA-MB-231 cells that were GLO1-depleted …
Figure 3—figure supplement 1
High endogenous MG induces YAP localization in breast cancer cells.
(A) Detection of MG was performed using MBo-specific fluorescent probe, as described in 'Materials and methods' section, and showed MG cellular increase in MDA-MB-468 cells that were GLO1-depleted …
Figure 3—figure supplement 2
High endogenous MG induces TAZ localization in breast cancer cells.
TAZ immunofluorescence staining in the indicated breast cancer cell line cultured in low- and high-glucose medium. Magnification 630x. Data are representative of two independent experiments.
Figure 3—figure supplement 3
Inhibition of glycolysis by treatment with 2-Deoxyglucose (2-DG) reverses YAP accumulation in MDA-MB-231 and MDA-MB-468 cells cultured in high-glucose medium.
(A and B) 2-DG (5 mM, 48 hr) significantly decreased lactate level measured using 1H-NMR and MG production quantified using FACS MBo mean fluorescence intensity (MFI) in highly glycolytic MDA-MB-231 …
Figure 3—figure supplement 4
Carnosine and aminoguanidine MG scavengers reverse YAP accumulation in MDA-MB-231 cells cultured in high-glucose medium.
MDA-MB-231 cells cultured in high-glucose condition until they reached high density and treated with carnosine (10 mM) or aminoguanidine (10 mM) impeded cellular accumulation of YAP. Magnification …
Figure 4 with 1 supplement
YAP cellular accumulation in shGLO1 MDA-MB-231 clones.
(A) GLO1 mRNA, (B) protein and (C) activity level in MDA-MB-231 shNT control and shGLO1#1 and #2. (D) YAP immunofluorescence (Santa Cruz antibody, H125) in MDA-MB-231 silenced for GLO1 (shGLO1#1 and …
Figure 4—figure supplement 1
Carnosine and aminoguanidine MG scavengers reverse YAP accumulation in GLO1-depleted MDA-MB-231.
(A) TAZ immunofluorescence staining in GLO1 depleted MDA-MB-231 cells. Magnification 630x. Data are representative of two independent experiments. (B) MDA-MB-231 silenced for GLO1 (shGLO1#1 and #2) …
Figure 5 with 3 supplements
MG induces YAP co-transcriptional activity in breast cancer cells.
(A) Stable knockdown of GLO1 (shGLO1#2) in MDA-MB-231 results in upregulation of several YAP target genes (ANKFN1, RIMS3, KCNK1, EMP2, OSBP2, IRAK3, WTN5A and CTGF) at the mRNA level as assessed by …
Figure 5—figure supplement 1
Inverse correlation between GLO1 and YAP target genes expression.
(A) Validation of YAP silencing (siYAP#1 and #2) in shGLO1 MDA-MB-231 cells 48 hr post-transfection by qRT-PCR. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as …
Figure 5—figure supplement 2
MG induces YAP co-transcriptional activity in breast cancer cells.
(A) Western blot detection of YAP in MDA-MB-231 cells under the indication conditions. Immunoblot is representative of three independent experiments. (B) Western blot of Phospho-Smad2/3 and Smad2/3 …
Figure 5—figure supplement 3
MG increases YAP-mediated migratory potential in breast cancer cells.
(A) E-cadherin and vimentin EMT markers are down-regulated and up-regulated, respectively, upon MG treatment in MDA-MB-468 breast cancer cells as shown by western blot. β-actin is used for …
Figure 6 with 1 supplement
MG induces LATS1 kinase decrease in breast cancer cells.
(A) Schematic representation of the Hippo pathway focused on MST1/2 and LATS1/2 kinases control of nuclear-cytoplasmic shuttling of YAP co-transcription factor. (B) LATS1, LATS2, MST1 and MST2 …
Figure 6—figure supplement 1
MG leads to YAP cellular accumulation through LATS1 expression decrease.
(A) MG treatment from low to high density did not affect LATS1 mRNA levels as assessed by qRT-PCR in the indicated cell lines. Data were analyzed using unpaired student’s t test and shown as the …
Figure 7 with 1 supplement
MG induces Hsp90 post-translational glycation in breast cancer cells.
(A) Immunoprecipitation of MG adducts on MG-treated MDA-MB-231 cells (300 µM, 6 hr) using a specific anti-argpyrimidine monoclonal antibody. Mouse immunoglobulins (IgG) were used as control. Total …
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Figure 7—source data 1
MG modifications on human recombinant Hsp90.
Peptides identified by mass spectrometry of MG-Hsp90 enzymatic digests. Modification sites are bolded and underlined. CEL: Carboxyethyllysine.
- https://doi.org/10.7554/eLife.19375.021
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Figure 7—source data 2
MG modifications on endogenous Hsp90.
Peptides identified by mass spectrometry of argpyrimidine immunoprecipitate from MDA-MB-231 treated with MG (300 µM) during 6 hr. Modification sites are bolded and underlined.-
- https://doi.org/10.7554/eLife.19375.022
Figure 7—figure supplement 1
MG induces Hsp90 post-translational glycation.
(A) MG treatment from low to high density did not affect HSP90 mRNA levels as assessed by qRT-PCR in the indicated cell lines. Data were analyzed using unpaired student’s t test and shown as the …
Figure 8 with 1 supplement
GLO1-depleted breast cancer cells show an increased tumorigenic potential in vivo.
(A) MDA-MB-231 shGLO1#1 and #2 and control shNT cells were injected subcutaneously in NOD-SCID mice (15 mice/group). After 4 weeks, primary tumors were surgically removed and weighed. Tumor weight …
Figure 8—figure supplement 1
GLO1-depleted breast cancer cells show an increased tumorigenic potential in vivo.
Validation of YAP silencing in GLO1-depleted MDA-MB-231 cells at different time points during tumor development on chicken chorioallantoic membrane described in Figure 8F–I.
Figure 9
GLO1-depleted breast cancer cells show an increased metastatic potential in a mouse xenograft model.
(A) MDA-MB-231 shGLO1#1 and #2 and control shNT cells were injected subcutaneously in NOD-SCID mice. After 4 weeks, primary tumors were surgically removed. Six weeks after tumor removal, mice were …
Author response image 1
Intracellular (A) and extracellular (B) MG concentrations in MDA-MB-231 cells.
MG adducts western blotting detection using anti-argpyrimidine antibody in MDA-MB-231 cells cultured in high and low glucose and upon exogenous MG treatment (C) and in Glo1-silenced cells (D).
Additional files
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Supplementary file 1
Antibodies and dilutions used for Western Blot experiments.
- https://doi.org/10.7554/eLife.19375.027
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Supplementary file 2
siRNA sequences.
- https://doi.org/10.7554/eLife.19375.028
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Supplementary file 3
Primer sequences and probes used for quantitative reverse transcription-PCR (qRT-PCR).
- https://doi.org/10.7554/eLife.19375.029
