Additional file 2: of Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer

Figure S1. RNA sequencing analysis of GLO1-depleted MDA-MB-231 cells. (A) MG extracellular concentrations were assessed over 48 h in conditioned medium of GLO1-depleted MDA-MB-231 cells using UPLC-MS/MS. (B) Volcano plots highlighting differentially expressed genes in shGLO1#1 and shGLO1#2 MDA-MB-231 cells. Orange and red dots represent genes differentially expressed significantly (q < 0.05 and log2 fold change (FC) > 1) for shGLO1 clones. Red dots represent genes of the pro-metastatic signature. (C) Tenascin C, Lumican and CD24 mRNA levels were assessed by qRT-PCR in MDA-MB-231 cells treated with MG 300 and 500 μM for 1 h. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S2. Dicarbonyl stress promotes anchorage-independent growth and invasion of breast cancer cells. Representative pictures of the colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-231 cells treated with carnosine. Figure S3. Dicarbonyl stress promotes invasion of breast cancer cells. (A) Migration ability toward serum of GLO1-overexpressing shNT and shGLO1#1 MDA-MB-231 cells was assessed using Transwell filters. Representative filters are shown for each condition. (B) Quantification of migration assays of MDA-MB-231 shGLO1 cells upon GLO1 overexpression. (C) GLO1 protein level was assessed using immunoblot in GLO1-silenced MDA-MB-231 upon GLO1 overexpression. β-actin protein is used as loading control. Western blot is representative of three independent experiments. (D) Invasiveness ability of GLO1-depleted MDA-MB-231 toward serum was assessed using Transwell filters. Where indicated, cells were pre-treated with MG scavengers, carnosine and aminoguanidine, 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. (E) Quantification of invasiveness ability of GLO1-silenced MDA-MB-231 cells treated with carnosine and aminoguanidine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S4. Highly migratory MCF7 cells (MCF7-M) display enhanced aerobic glycolysis. (A) Representative pictures of cultured MCF7 and MCF7-M cells. Scale bar represents 400 μm. (B) Extracellular acidification rates (ECAR) in MCF7 and MCF7-M cells using Seahorse flux analyzer. Glycolytic-related ECAR was calculated. Data were analyzed using Student’s t test and shown as mean values ± SEM of two independent experiments. **p < 0.01. Figure S5. Dicarbonyl stress promotes migration and anchorage-independent growth of MDA-MB-468 breast cancer cells. (A) GLO1 protein level in MDA-MB-468 shNT control and shGLO1#2 and #3 cells. β-actin protein is used as loading control. Western blot is representative of three independent experiments. (B) Lumican and CD24 mRNA levels were assessed by qRT-PCR in GLO1-depleted MDA-MB-468 cells. (C) Lumican and CD24 mRNA levels were assessed by qRT-PCR in MDA-MB-468 cells treated with MG 300 and 500 μM for 1 h. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. (D) Migration ability of GLO1-depleted MDA-MB-468 toward serum was assessed using Transwell filters. Where indicated, cells were pre-treated with carnosine 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. (E) Quantification of migratory ability of GLO1-silenced MDA-MB-468 cells treated with carnosine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. (F) Representative pictures of the colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-468 cells treated with carnosine. (G) Quantification of colonies formed in a soft agar matrix by GLO1-silenced MDA-MB-468 cells treated with carnosine. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S6. P-ERK localization in GLO1-depleted breast cancer cells. P-ERK immunofluorescence staining in GLO1-silenced MDA-MB-231 (A) and MDA-MB-468 (B) cells cultured in serum-free conditions. Data are representative of three independent experiments. Magnification × 630. Zoomed pictures (white square) are shown where indicated. Figure S7. GLO1 rescue impairs increased SMAD1 phosphorylation in GLO1-depleted MDA-MB-231 cells and SMAD1 activation favors the enhanced migration ability of MCF7-M cells. (A) (P-)SMAD1 (ser206) protein levels were assessed using immunoblot in shNT and shGLO1#1 MDA-MB-231 cells upon GLO1 overexpression. (B) P-SMAD1 (ser206) and SMAD1 protein level in MCF7 and MCF7-M cells cultured in serum-free conditions. (C) Migration ability toward serum of SMAD1-silenced MCF7 and MCF7-M cells was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. (D) Quantification of migration assays of SMAD1-silenced MCF7 and MCF7-M cells. (E) SMAD1 protein level assessed by immunoblot to validate SMAD1-silencing using siRNAs in MCF7 and MCF7-M cells (related to panels B and C). β-actin or HSC70 were used as loading control. Immunoblots were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. All western blots are representative of three independent experiments. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S8. SMAD1 activation promotes the pro-metastatic phenotype in MDA-MB-468 cells. A. SMAD1 target genes mRNA levels were assessed using qRT-PCR in SMAD1-silenced MDA-MB-468 shGLO1 cells. SMAD1 mRNA level was assessed to validate efficient SMAD1 siRNA silencing. (B) Migration ability toward serum of SMAD1-silenced MDA-MB-468 shGLO1 cells was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. (C) Quantification of migration assays of SMAD1-silenced MDA-MB-468 shGLO1 cells. (D) SMAD1 protein level assessed by immunoblot to validate SMAD1-silencing using siRNAs in GLO1-depleted MDA-MB-468 cells (related to panels B and C). Western blots are representative of three independent experiments. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S9. GLO1 depletion is associated with a global decrease of phosphatases expression in breast cancer cells. (A) mRNA levels of PPP2 phosphatases (from catalytic, structural and regulatory subunits) in GLO1-depleted MDA-MB-231 and MDA-MB-468 cells were assessed by qRT-PCR. (B) DUSP1, 5, 8, 10, 12, 14 and 16 phosphatases mRNA levels in GLO1-depleted MDA-MB-468 cells were quantified by qRT-PCR. Data were analyzed using one-way ANOVA followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. Figure S10. DUSP5 overexpression decreases the migratory capacity of GLO1-depleted breast cancer cells. Migration ability toward serum of DUSP5-overexpressing MDA-MB-231 (A) and MDA-MB-468 (B) cells was assessed using Transwell filters. Representative filters are shown for each condition. Table S1. Remarkable genes coding for ECM components and ECM regulators, the expression of which is significantly modulated in GLO1-depleted MDA-MB-231 breast cancer cells. Genes shown in bold have been validated at the protein and/or mRNA expression levels. ns, not significant. Table S2. DUSPs and PPPs subunits expression data from RNASeq in GLO1-depleted MDA-MB-231 cells. Fold change and false discovery rate (FDR) are shown for both shGLO1 clones. ns, not significant. Table S3. Primer sequences and probes used for quantitative reverse transcription-PCR (qRT-PCR). Table S4. Antibodies and dilution used for western blot experiments. (PDF 2523 kb)