Altered propionate metabolism contributes to tumour progression and aggressiveness

Abstract

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl coenzyme A epimerase (MCEE), mediated by an extracellular signal-regulated kinase 2-driven transcription factor Sp1/early growth response protein 1 transcriptional switch driven by metastatic signalling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and intracellular and intratumoral accumulation of methylmalonic acid, a by-product of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.

Extended Data Fig. 1. Methylmalonic acid and MCEE levels are altered by metastatic signaling in different cancer cell models.

(a) Propionate metabolism-related enzyme levels evaluated by immunoblots in 4T1-derived clones of cells with different metastatic potential; representative images (n=4). (b) MMA levels in A549 cells treated with TGFβ + TNFα for 3 days (n=4, two-tailed t-test). c, Propionate metabolism-related enzyme levels evaluated by immunoblots in A549 cells treated with TGFβ + TNFα for 3 days; representative images (n=4). d, MCEE-luciferase promoter activity in A549 cells treated with TGFβ + TNFα for 3 days (n=4, two-tailed t-test). e, MMA levels in non-metastatic and metastatic triple negative breast cancer human cell lines (n=4). f, Kaplan-Meyer survival curve of breast cancer patients as a function of MCEE expression. g, Kaplan-Meyer survival curve of lymph node positive triple negative breast cancer patients as a function of MCEE expression. All values are expressed as mean ± SEM.

Extended Data Fig. 2. Knockdown of MCEE induces a pro-aggressive reprogramming.

a, b, MMA levels in HCC1806 (a) and MCF-10A (b) cells with MCEE knockdown for 2 days (n=4, one-way ANOVA with Tukey’s multiple comparison test). c, Immunoblots for EMT and aggressiveness markers in HCC1806, MCF-10A and A549 cells with MCEE knockdown for 10 days; representative images (n=4). All values are expressed as mean ± SEM.

Extended Data Fig. 3. Suppression of MUT induces a pro-aggressive reprogramming.

a, MMA levels in MCF-10A cell with MUT knockdown for 3 days (n=3, one-way ANOVA with Tukey’s multiple comparison test). b, c, Immunoblots for EMT and aggressiveness markers in MCF-10A (b) and A549 (c) cells with MUT knockdown for 10 days; representative images (n=4). d, e, f, g, mRNA levels of SOX4 (d), TGFB1 (e), TGFBR1 (f), and TGFBR3 (g) evaluated by RNA sequencing in A549 cells with MUT knockdown for 3 days (n=3, one-way ANOVA with Tukey’s multiple comparison test). h, MMA levels in MDA-MB-231-LM2 versus MDA-MB-231-luciferase parental cells (n=8, two-tailed t-test). All values are expressed as mean ± SEM.

Extended Data Fig. 4. Vitamin B12 deficiency induces a pro-aggressive reprogramming.

a, MMA levels in MCF-10A cells grown in complete or Vitamin B12-depleted media for 9 days (n=3, two-tailed t-test). b, Immunoblots for EMT and aggressiveness markers in HCC1806, MCF-10A and A549 cells grown in complete or Vitamin B12-depleted media for 10 days; representative images (n=4). c, d, MMA levels in HCC1806 (n=4) (c) and MCF-10A (n=4) (d) cells with MMAB knockdown for 3 days (one-way ANOVA with Tukey’s multiple comparison test). e, Immunoblots for EMT and aggressiveness markers in HCC1806, MCF-10A and A549 cells with MMAB knockdown for 10 days; representative images (n=4). All values are expressed as mean ± SEM.

Extended Data Fig. 5. Overexpression of PCC induces a pro-aggressive reprogramming.

a, b, Propionyl-CoA (a) and MMA (b) levels in MCF-10A cells overexpressing PCCA and PCCB for 5 days (n=3, two-tailed t-test). c-f, TCA cycle intermediates succinate (c), fumarate (d), malate (e), oxaloacetate (f) in MCF-10A cells overexpressing PCCA and PCCB for 5 days (n=3, two-tailed t-test). g, Immunoblots for EMT and aggressiveness markers in HCC1806, MCF-10A and A549 cells overexpressing PCCA and PCCB for 10 days; representative images (n=4). h, i, Transwell migration (h) and invasion (i) assays of MDA-MB-231-luciferase parental cells overexpressing PCCA and PCCB for 6 days (n=4, two-tailed t-test). j, k, Lung colonization assay of MDA-MB-231-luciferase parental cells injected after 6 days of PCCA and PCCB overexpression, imaged at 6 weeks; representative images (j) and quantification (k) (n=10, two-tailed t-test). All values are expressed as mean ± SEM.

Extended Data Fig. 6. Knockdown of PCCA does not induce EMT.

a, b, Immunoblots for EMT markers in MCF-10A (a), and A549 (b) cells with PCCA knockdown for 10 days; representative images (n=4). c, Immunoblots for EMT markers in MCF-10A and A549 cells with PCCA knockdown and treated with 5 mM MMA for 10 days; representative images (n=4). d, Immunoblots for EMT markers in A549 cells with PCCA knockdown and treated with TGFβ + TNFα for 5 days; representative images (n=4). e, MMA levels in Hs578T cells with PCCA knockdown for 5 days (n=4, one-way ANOVA with Tukey’s multiple comparison test). f, g, Transwell migration (f) and invasion (g) assays of Hs578T with PCCA knockdown for 5 days (n=4, one-way ANOVA with Tukey’s multiple comparison test). h, i, Proliferation of Hs578T (h) and MDA-MB-231-LM2 (i) with PCCA knockdown for 5 days (n=4, two-way repeated measures ANOVA test based on general linear model (GLM) with Tukey’s multiple comparison test, p values only shown for end point). All values are expressed as mean ± SEM.

Fig. 1:. Methylmalonic Acid is upregulated in breast cancer metastasis.

a, b, Heat map (a) and metabolic pathway enrichment analysis (b) of the statistically significantly altered metabolites (FDR≤0.05) in 4T1 primary tumors and pulmonary metastases (n=4 biologically independent samples). c, Schematic representation of propionate metabolism. d, Methylmalonic acid (MMA) levels in 4T1 primary tumors and pulmonary metastases (n=4, two-tailed t-test). e, MMA levels in 4T1 (broadly metastatic) and 4TO7 (locally invasive) clones with different metastatic potential derived from a single primary tumor (n=5 biologically independent samples, two-tailed t-test). f, MMA levels in breast epithelial and breast cancer cell lines (n=5 biologically independent samples). All values are expressed as mean ± SEM.

Fig. 2:. Metastatic signaling leads to MMA production through regulation of MCEE.

a, b, MMA levels in MCF-10A (a) and HCC1806 (b) cells treated with TGFβ + TNFα for 3 days (n=4 biologically independent samples, two-tailed t-test). c, Fractions of labeled intracellular MMA derived from glucose + glutamine (GG) or valine + isoleucine + threonine + methionine (AA) in HCC1806 cells treated with TGFβ + TNFα for 3 days (n=6 biologically independent samples, two-way ANOVA with Sidak’s multiple comparison test). d, e, Propionate metabolism-related enzyme levels evaluated by immunoblots in MCF-10A and HCC1806 cells treated with TGFβ + TNFα for 3 days (d), and in non-metastatic and metastatic triple negative breast cancer human cell lines (e); representative images (n=4 biologically independent samples). f, g, MCEE mRNA levels evaluated by qPCR in MCF-10A (f) and HCC1806 (g) cells treated with TGFβ + TNFα for 3 days (n=4 biologically independent samples, two-tailed t-test). h, MCEE-luciferase promoter activity in MCF-10A cells treated with TGFβ + TNFα for 3 days (n=4 biologically independent samples, two-tailed t-test). i, Schematic representation of SP1 and EGR1 binding sites in MCEE promoter. j, Propionate metabolism-related enzyme levels evaluated by immunoblots in MCF-10A cells expressing the metastatic-inducer ERK2 D319N mutant for 3 days; representative images (n=4 biologically independent samples). k, MMA levels in MCF-10A cells expressing the metastatic-inducer ERK2 D319N mutant for 3 days (n=4 biologically independent samples, two-tailed t-test). l, MCEE-luciferase promoter activity in MCF-10A cells expressing the metastatic-inducer ERK2 D319N mutant for 3 days (n=4 biologically independent samples, two-tailed t-test). m, n, MCEE protein levels evaluated by immunoblot in MCF-10A and HCC1806 cells with SP1 knockdown for 3 days (m) and in MCF-10A cells expressing the ERK2 D319N mutant and either SP1 wild-type or the SP1 T453/T739 phosphorylation site mutants (S to A phospho-defective mutant; S to E, phospho-mimetic mutant) for 3 days (n); representative images (n=4 biologically independent samples). o, MCEE, EGR1 and phospho SP1 protein levels evaluated by immunoblot in HCC1806 treated with TGFβ + TNFα or TGFβ + TNFα + MEK inhibitor for 3 days; representative images (n=4 biologically independent samples). All values are expressed as mean ± SEM.

Fig. 3:. Intracellular MMA production promotes EMT and aggressive properties.

a, b, MMA levels (one-way ANOVA with Tukey’s multiple comparison test) (a) and immunoblots for EMT and aggressiveness markers (b) in HCC1806 cells with MUT knockdown for 3 days; representative images (n=4 biologically independent samples). c, Functional annotation clustering analysis of mRNAs that changed >1.5-fold when evaluated by RNA sequencing in A549 cells with MUT knockdown for 3 days (n=3 biologically independent samples). d, MMA levels in MDA-MB-231-luciferase parental cells with MUT knockdown for 3 days (n=4 biologically independent samples, one-way ANOVA with Tukey’s multiple comparison test). e, f, Transwell migration (e) or invasion (f) assays of MDA-MB-231-luciferase parental cells with knockdown of MUT for 6 days (n=4 biologically independent samples, one-way ANOVA with Tukey’s multiple comparison test). g, h, Lung colonization assay of MDA-MB-231-luciferase parental cells injected after 6 days of MUT knockdown imaged at 6 weeks; representative images (g) and quantification (h) (n=8 biologically independent animals for shNT and shMUT#2 and n=7 for shMUT#1, one-way ANOVA with Tukey’s multiple comparison test). All values are expressed as mean ± SEM.

Fig. 4:. PCC regulates MMA levels and determines pro-aggressive properties.

a, MMA levels in MCF-10A cells with PCCA knockdown and treated with TGFβ + TNFα for 3 days (n=4 biologically independent samples, two-way ANOVA with Sidak’s multiple comparison test). b, EMT-related proteins evaluated by immunoblots in MCF-10A cells with PCCA knockdown and treated with TGFβ + TNFα for 5 days; representative images (n=4 biologically independent samples). c, MMA levels in MDA-MB-231-LM2 cells with PCCA knockdown for 5 days (n=4 biologically independent samples, one-way ANOVA with Tukey’s multiple comparison test). d, Mesenchymal protein levels evaluated by immunoblots in MDA-MB-231-LM2 cells with PCCA knockdown and treated with 5 mM MMA for 5 days; representative images (n=4 biologically independent samples). e, f, Transwell migration (e) or invasion (f) assays of MDA-MB-231-luciferase LM2 cells with knockdown of PCCA for 6 days (n=4 biologically independent samples, one-way ANOVA with Tukey’s multiple comparison test). g, h, Lung colonization assay of MDA-MB-231-luciferase LM2 cells with knockdown of PCCA for 6 days; representative images (g) and quantification (h) (n=10 biologically independent animals, one-way ANOVA with Tukey’s multiple comparison test). All values are expressed as mean ± SEM.