Abstract
Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine–sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvβ3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness.
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Data availability
All data supporting the findings of this study are available within the Article and the Supplementary Information, and from the corresponding author on reasonable request. Gel source images are available in Supplementary Fig. 1. Transcriptomic data are available at the GEO under accession code GSE198380. Source data are provided with this paper.
Code availability
All the custom code used in the study is available from the corresponding author on reasonable request.
Change history
30 August 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41586-022-05226-7
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Acknowledgements
We thank M. Vander Heiden (MIT) for providing the PHGDH overexpression plasmid; P. Bieniasz-Krzywiec (VIB-KU Leuven) for his help with the Transwell migration assay and V. van Hoef (VIB Bioinformatics Core Facility) for his advice on the RNA-seq analysis; and Raze Therapeutics for providing us with the PHGDH inhibitor PH-755. The breast tissue and data bank at the Goodman Cancer Research Institute of McGill University Health Centre (MUHC) is supported by the Database and Tissue Bank Axis of the Réseau de Recherche en Cancer of the Fonds de Recherche du Québec-Santé and the Québec Breast Cancer Foundation and certified by the Canadian Tumor Repository Network (CTRNet). The illustrations in Fig. 4e and Extended Data Fig. 3g were created using BioRender.com. M.R. has received consecutive postdoctoral fellowships from the FWO and Stichting tegen Kanker and an Early Access Grant from the VIB Technology Watch Team Program. P.A.-M. has received funding from Marie Curie Actions and the Beug Foundation. A.M.C. has received a fellowship from Boehringer Ingelheim Fonds. G.D. and G.R. have received consecutive PhD fellowships from Kom op tegen Kanker and the FWO, and A.V. from the FWO. H.F.A. has received a fellowship from the Stichting tegen Kanker. J.F.-G. has received consecutive postdoctoral fellowships from the FWO. S.P. is a VIB international scholar. J.v.R. and L.B. were funded by Cancer Genomics Netherlands and Doctor Josef Steiner Foundation. T.G.P.G. acknowledges funding from the Barbara and Wilfried Mohr Foundation, the Matthias-Lackas Foundation, the Dr Leopold and Carmen Ellinger Foundation, the Dr Rolf M. Schwiete Foundation, the German Cancer Aid (DKH-70112257, DKH-70114111), the Gert and Susanna Mayer Foundation, the Boehringer Ingelheim Foundation and the SMARCB1 association. P.C. is supported by Grants from Methusalem funding (Flemish government) and the NNF Laureate Research Grant from Novo Nordisk Foundation (Denmark). S.Y.L. was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA270136 and the METAvivor Early Career Investigator Grant. G.J.H. is supported by Cancer Research UK (C9545/A29580) and is a Wolfson Royal Society Research Professor (RP130039 and RSRP\R\200001). M. Park is grateful for the support from the Quebec Cancer Consortium and the financial support from the Ministère de l’Économie et de l’Innovation du Québec through the Fonds d’accélérations des collaborations en santé. The Patient-derived Xenograft and Advanced In Vivo Models Core at Baylor College received funding from CPRIT Core Facility Award (RP170691) and P30 Cancer Center Support Grant (NCI-CA125123). S.-M.F. acknowledges funding from the European Research Council under the ERC Consolidator Grant Agreement no. 771486, MetaRegulation; FWO, research projects (G088318N); KU Leuven, FTBO, King Baudouin Foundation, Beug Foundation and Fonds Baillet Latour.
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Contributions
M.R., P.A.-M., M.D. and G.D. performed most of the experiments, and M.R. and P.A.-M. analysed all data. M.F., D.Z., M. Park and D.N. helped with multiplex IHC, and C.J. and G.J.H. performed IMC analysis. L.B. performed and J.v.R. supervised intravital imaging. A.V., G.D. and M. Planque performed metabolomics experiments. D.B., I.V., J.V.E. and G.D. helped with in vivo experiments. H.-F.A. and D.N. helped with microscopy and IHC analysis. J.F.-G., T.V.B. and S.D. performed bioinformatics analysis. A.M.C. helped with CRISPR and overexpression construct designs and cell line generation. S.P., G.R., F.R., A.A.P. and L.A.B. helped with in vitro experiments. A.B.A. and P.K. performed, and S.J.M. and J.-C.M. supervised the experiments with the melanoma model. T.G.P.G., K.S., H.B., M.M.L.K. and M.F.O. provided human samples and their analysis. L.E.D. and M.T.L. performed PDX experiments. P.v.V. performed proteomics analysis. A.B., T.V.B. and D.L. performed bulk and single-cell RNA-seq analysis. C.R.-D., T.Z., S.T.T., G.E., M.W., P.C., J.C., M.M. and S.Y.L. provided reagents, methods and expertise. S.-M.F. designed the study and wrote the manuscript. S.-M.F. conceived and supervised the study and obtained funding.
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Competing interests
S.-M.F. has received funding from Bayer AG, Merck, Alesta Therapeutics and Black Belt Therapeutics, has consulted for Fund+ and is in the advisory board of Alesta Therapeutics. T.G.P.G. has consulted for Boehringer Ingelheim. M.T.L. is an uncompensated president, CEO and limited partner of StemMed and an uncompensated manager in StemMed Holdings LP, its general partner; a founder and equity holder in Tvardi Therapeutics; and a faculty member at Baylor College of Medicine. L.E.D. is a compensated employee at Baylor College of Medicine. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Circulating tumour cells and early metastatic lesions exhibit low PHGDH expression.
a. Distribution of PHGDH expression in human TNBC primary tumour specimens. PHGDH expression was assessed by immunohistochemistry (n = 129). b. Comparison of the tumour grade in human TNBC primary tumours with homogeneous high and heterogeneous/low PHGDH expression (n = 126). Chi-squared test. c. Comparison of the tumour stage (pT) in human TNBC primary tumours with homogeneous high and heterogeneous/low PHGDH expression (n = 129). Chi-squared test. d. Comparison of the lymph node stage (pN) in human TNBC primary tumours with homogeneous high and heterogeneous/low PHGDH expression (n = 128). Chi-squared test. e. Metastasis occurrence in patients with TNBC bearing primary tumours with homogeneous high (17 out of 87) or heterogeneous/low (14 out of 42) PHGDH expression (n = 129). Fisher’s exact test, two-sided. f. Kaplan-Mayer curve comparing the percentage of survival of patients with TNBC with heterogeneous/low and homogeneous PHGDH protein expression in the primary tumour (n = 129). Mantel-Cox test. g. Representative picture of PHGDH protein heterogeneity in the primary tumour from orthotopic (mammary fat pad, m.f.p.) 4T1 mouse model, assessed by immunohistochemistry. Green, PHGDH; blue, DAPI nuclear staining. Scale bar 1 mm. h. Representative picture of PHGDH protein heterogeneity in the primary tumour from orthotopic (m.f.p.) TNBC PDX model, assessed by immunohistochemistry. Green, PHGDH; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 200 μm. i. Distribution and correlation of PHGDH and Ki67 protein expression in the primary tumour from orthotopic (m.f.p.) 4T1 mouse model assessed by imaging mass cytometry. Nonparametric Spearman rank correlation, two-tailed. Scale bar 400 μm. j, k. Correlation of PHGDH and phospho-histone H3 (PHH3) protein expression in the primary tumour from orthotopic (m.f.p.) TNBC PDX model assessed by immunohistochemistry (j). The pooled analysis of 3 different PDX models, on 9 randomly chosen microscopy fields for each model, is shown in (k). Nonparametric Spearman rank correlation, two-tailed. l. Correlation plot between GSVA-based Z-scores for a gene expression signature indicative of low PHGDH and one indicative of EMT, with the colour code indicating Ki67 normalized expression levels (CP100k = counts per 100 k reads), based on single-cell RNA-seq data for primary tumours of 13 patients with TNBC. m. Representative images of the expression levels of PHGDH protein in circulating tumour cells (CTCs) compared to the respective primary tumours from TNBC PDX models, assessed by immunohistochemistry. Green, PHGDH; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 25 μm. n. Expression levels of PHGDH protein in circulating tumour cells (CTCs) compared to the corresponding primary tumours from TNBC PDX models, assessed by immunohistochemistry (complementary to Fig. 1c). Analysis performed on PDX models BCM-3611-R3TG4 (7 mice, 35 randomly chosen microscopy fields for the primary tumours, 5 per mouse, 104 single CTCs) and BCM-4272-R3TG6 (7 mice, 35 randomly chosen microscopy fields for the primary tumours, 5 per mouse, 101 single CTCs). The solid lines indicate the median, the whiskers indicate the 95% confidence interval. Unpaired t test with Welch’s correction, two-tailed
Extended Data Fig. 2 PHGDH protein levels but not mRNA expression is low in early metastatic lesions compared to primary tumours and advanced metastatic lesions.
a, b. Western blot of PHGDH in lungs, primary breast tumours and lung metastases from orthotopic (m.f.p.) 4T1 (n = 4) (a) and EMT6.5 (n = 4) (b) mouse models. c. Western blot analysis of PHGDH in primary tumours and liver metastases from two different orthotopic (subcutaneous) melanoma PDX models (n = 4). d. Positivity to PHGDH in primary breast tumours (n = 9) and early lung metastases (~16 weeks after primary tumour initiation; n = 52) from TNBC PDX models assessed by immunohistochemistry. e. Representative images of PHGDH protein expression in lymph node metastases and matching primary breast tumours from patients with TNBC assessed by immunohistochemistry. Green, PHGDH; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 50 μm. f. Relative change in Phgdh gene expression in lungs, primary breast tumours and lung metastases from orthotopic (m.f.p.) 4T1 and EMT6.5 mouse models (n = 4). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison. g. Western blot of PHGDH in lung metastases from orthotopic (m.f.p.) 4T1 mouse model (n = 4), at 3, 4 and 5 weeks after injection of the cancer cells. h. Representative pictures of PHGDH protein expression in primary and metastatic melanoma mouse model (Tyr::N-Ras+/Q61K;Ink4a−/−). Left panels represent tumours from mice injected with melanoma cells alone; middle and right panels represent tumors from mice co-injected with melanoma and Bend3 endothelial cells (ratio 1:4). Green, Phgdh; red, dsRed tumour cell marker; blue, DAPI nuclear staining. i. Western blot of PHGDH levels in 4T1 cells upon hypoxia and reoxygenation, serine pull-out compared to full medium, and treatment with salubrinal (ATF4 activation) or thapsigargin (ER stress induction). One representative experiment is shown (n = 3). j. Representative pictures of expression levels of PHGDH and ATF4 activation markers in TNBC PDX model, assessed by multiplex immunofluorescence. Turquoise, p-GCN2; White, ATF4; Pink, pS6; Green, PHGDH; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 200 μm
Extended Data Fig. 3 Proximity to endothelial cells induces loss of PHGDH in cancer cells.
a, b. PHGDH expression in 4T1 (1:2 and 1:5 ratio) and EMT6.5 cells (1:2 ratio) co-cultured with Bend3 immortalized mouse endothelial cells based on immunofluorescence. Left panel, representative pictures (scale bar 50 μm), red, PHGDH; blue, DAPI nuclear staining. The image represented was selected to show only 4T1/EMT6.5 cells based on pan-cytokeratin expression in cancer cells and GFP expression in Bend3 cells. Right panel, fluorescent intensity quantification (n = 4 independent samples). Solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison (a) and unpaired t test with Welch’s correction, two-tailed (b). c. Venn diagram depicting the number of overlapping enriched gene sets, based on GSEA in RNA-seq data from 4T1 Phgdh knockdown (shPHGDH) compared to control cells (left), versus enriched gene sets in 4T1 and EMT6.5 cells co-cultured (1:2 ratio) with Bend3 immortalized mouse endothelial cells (EC Co-Culture) compared to mono-cultured 4T1 or EMT6.5 cells (right). The numbers at the top represent the total enriched gene-sets found in the corresponding dataset. d. Lung metastatic area in mice co-injected with 4T1 cancer cells and Bend3 endothelial cells (ratio 1:2) or 4T1 cells alone in the mammary fat pad (n ≥ 10). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Unpaired t test with Welch’s correction, two-tailed. e. PHGDH protein expression in 4T1 primary tumour of mice injected with cancer cells alone or co-injected with Bend3 immortalized mouse endothelial cells assessed by immunohistochemistry. Green, Phgdh; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 200 μm. f. Representative pictures of PHGDH protein expression in primary melanoma model (Tyr::N-Ras+/Q61K;Ink4a−/−) from mice injected with melanoma cells alone or co-injected with melanoma and Bend3 endothelial cells (ratio 1:4). Outcome of this experiment is also depicted in Extended Data Fig 2h. Upper panel represents green and red channels of the same tumor region as the panel on the top left in Extended Data Fig. 2h. Lower panel represents green and red channels of the same tumour region as the panel on the top right in Extended Data Fig. 2h. Green, Phgdh; red, dsRed tumour cell marker; blue, DAPI nuclear staining. Scale bar 200 μm. g. Schematic representation of the time-lapse intravital imaging experiment setup. h. Track length of migratory 4T1 shSCR-mTurquoise and 4T1 shPHGDH-Dendra in primary tumours from orthotopic (m.f.p.) 4T1 mouse model assessed by time-lapse intravital imaging. The solid lines indicate the median, the dashed lines indicate the 25th and 75th percentiles. Unpaired t test with Welch’s correction, two-tailed. i. Rate of metastatic progression of lung metastases from mice injected with either 4T1 shSCR (n = 10 per time point) or 4T1 shPHGDH cells (n = 10 per time point). Error bars represent standard deviation (s.d.) from mean. Unpaired t test with Welch’s correction, two-tailed. j, k. Invasive capacity of 4T1 and EMT6.5 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells in a 3D matrix. Invasion was assessed by measuring the invasive area of cancer cells stained with calcein green. Representative images are depicted in the left panel (scale bar 500 μm), quantification in the right panel. Each dot represents a different, randomly selected microscopy field (n = 5 for (j), n = 10 for (k)). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison (j) and Unpaired t test with Welch’s correction, two-tailed (k)
Extended Data Fig. 4 Loss of PHGDH induces a partial EMT and the expression of markers indicating altered cell-cell or cell matrix interactions in cancer cells.
a. Migratory ability of 4T1 and EMT6.5 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells. Migration was assessed in plain transwells and in transwells coated with either vascular endothelial cells (HUVECs) or lymphatic endothelial cells (LECs) (n = 3). Two-way ANOVA.b, c. Volcano plots showing genes (b) or proteins (c) upregulated or downregulated upon loss of PHGDH based on RNA-seq (b) or proteomics (c) data for 4T1 cells with Phgdh knockdown (shPHGDH) relative to control cells (shSCR). The cutoffs represent p-values of 0.01 and absolute fold-changes of 1.25. Genes (b) or proteins (c) indicative of EMT are shown in red, with labels included for select genes/proteins above the respective p-value/fold-change cutoffs. d. Relative change in Twist, vimentin (Vim) and Snai1 gene expression upon PHGDH knockdown in 4T1 and EMT6.5 cells. Error bars represent standard deviation (s.d.) from mean (n = 3). Unpaired t test with Welch’s correction, two-tailed. e. Western blot of phosphorylated proto-oncogene tyrosine-protein kinase Src (c-Src) and p38 mitogen-activated protein kinase upon Phgdh knockdown in 4T1 and EMT6.5 cells. One representative experiment is shown (n = 3). f. Western blot of Vimentin and Twist in 4T1 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR). One representative experiment is shown (n = 3). g. Western blot of phosphorylated proto-oncogene tyrosine-protein kinase Src (c-SRC) and p38 mitogen-activated protein kinase upon Phgdh overexpression in MDA-MB-231 cells. One representative experiment is shown (n = 3). h. Representative pictures of PHGDH and EMT markers protein expression in TNBC PDX model assessed by multiplex immunofluorescence. Green, PHGDH; Yellow, VIMENTIN; Pink, TWIST; Orange, E-CADHERIN; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 200 μm. i. Invasive area and distance of 4T1 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells (n = 15 independent samples). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Unpaired t test with Welch’s correction, two-tailed. j, k. Invasive capacity of 4T1 and MDA-MB-231 cells upon PHGDH overexpression (PHGDH OE) compared to control (CTR) cells in a 3D matrix. The invasive area of cancer cells was stained with calcein green. Representative images are depicted in the left panel (scale bar 500 μm), quantification in the right panel. Each dot represents a different microscopy field (n = 5). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Unpaired t test with Welch’s correction, two-tailed.
Extended Data Fig. 5 Modulation of PHDGH expression alters gene expression signatures related to metastasis formation.
a. GSEA showing the top 50 commonly upregulated gene sets upon integration of the RNA-seq/proteomics datasets for 4T1 cells upon Phgdh knockdown (shPHGDH) compared to control cells, and the RNA-seq dataset for 4T1 or EMT6.5 cells co-cultured with Bend3 immortalized mouse endothelial cells, compared to mono-cultured 4T1 or EMT6.5 cells. Normalized enrichment scores (NES) for each data set are indicated by the coloured symbols, as defined in the plot legend. Gene sets are ranked based on their average NES among all three data sets, indicated by the white dots, with those gene sets with the highest mean NES shown on top. The red dash-dotted line indicates a NES of 1. The gene-set entries on the y-axis include three single-sample signatures comprising the most differentially upregulated genes in each of the three data sets (colour-coded identically to the plot legend), as well as a signature consisting in the intersection of those for the Phgdh knockdown and co-culture RNA-seq data (colour-coded in orange), indicative of low Phgdh protein expression. The remaining entries on the y-axis are colour-coded based on their belonging to one of the following categories: EMT and ECM remodelling (pink), integrin signalling (burgundy), OPN/AP-1 signalling and MMP activity (light brown), or other (black). b. GSEA results based on RNAseq data for MDA-MB-231 cells upon PHGDH overexpression (PHGDH OE) compared to control cells for the identical gene sets as in a. The red dash-dotted lines indicate a NES of ±1, whereas the black dotted line indicates a NES of 0. Data points are colour-coded according to the same colour scheme used for the respective gene-set entries in a. c. Correlation plots of GSVA-derived Z-scores for 3 of the top 50 hits found upon integration of the RNA-seq/proteomics/co-culture datasets (see Extended Data Fig. 5a) versus the scores for the Hallmark EMT gene signature (on the x-axis). Data were obtained from RNA-seq of scRNA-seq data for primary tumours of 13 patients with TNBC. The colour code indicates the Z-Score for a gene expression signature indicative of low PHGDH protein expression. Total least-squares regression lines and confidence intervals are overlaid on top of each plot, with the corresponding Pearson correlation coefficient (R) values shown on the top-left corners. d. Activity of extracellularly secreted MMP-3 (μU/min) in cell culture media collected from invasion assays of 4T1 cells upon Phgdh knockdown (shPHGDH) or control (shSCR) cells after 72 h of seeding (n = 3 independent experiments). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. One-way ANOVA with Dunn’s multiple comparison. e. Invasive capacity of EMT6.5 cells pre-treated (24h) with an antibody against integrin αvβ3 or control IgG (2.5 μg ml−1) and upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells in a 3D matrix. The invasive area was stained with calcein green. Each dot represents a different microscopy field (n = 5). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison.
Extended Data Fig. 6 Low PHGDH protein expression increases sialic acid metabolism and promotes glycosylation.
a. Schematic representation of glycolysis and its branching metabolic pathways. Enzymes are depicted in bold, pathway names in italics. Solid lines represent single reactions, dashed lines recapitulate multiple reactions. b. Metabolite abundances of sialic acid, UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-sialic acid upon Phgdh knockdown in 4T1 cells (n = 15). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and the maximum values. Unpaired t test with Welch’s correction, two-tailed. c. Dynamic 13C6 glucose labelling of MDA-MB-231 cells showing 13C incorporation into sialic acid, UDP-N-acetylglucosamine and CMP-sialic acid upon PHGDH overexpression (PHGDH OE) (n=3). Error bars represent s.d. from mean. Two-way ANOVA. d. Metabolite abundances of sialic acid, UDP-N-acetylglucosamine and CMP-sialic acid upon PHGDH overexpression (PHGDH OE) in MDA-MB-231 cells (n = 15). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the smallest and the largest values. Unpaired t test with Welch’s correction, two-tailed. e. Sialic acid/GlcNAc-containing-proteins isolated from whole cell lysate of 4T1 cells upon Phgdh knockdown (shPHGDH) or control (shSCR) cells treated with tunicamycin (0.05 μg ml−1) or DMSO for 72 h. Total isolated Sialic acid/GlcNAc-linked proteins were quantifying using Qubit™ Protein Assay Kit (n = 2 independent experiments). Error bars represent s.d. from mean. f. Levels of β-1,4-GlcNAc- and sialic acid-linked residues in 4T1 Phgdh knockdown (shPHGDH) and control (shSCR) cells after 72 h of tunicamycin pretreatment (0.05 μg ml−1) measured at 7, 24 and 48 h after tunicamycin removal using wheat germ agglutinin (WGA) staining (n = 3). Red, WGA β-1,4-GlcNAc- and sialic acid-linked proteins; blue, DAPI nuclear staining. Error bars represent standard deviation (s.d.) from mean. Unpaired t test with Welch’s correction, two-tailed. Scale bar 20 μm. g. Cell viability upon tunicamycin (0.05 μg ml−1) and PHGDH inhibitor (PH755, 1 μM) treatment (36h) in 4T1 cells upon Phgdh knockdown (shPHGDH) or control (shSCR) (n = 5). Error bars represent s.d. from mean. One-way ANOVA with Turkey’s multiple comparison test. h. Protein expression levels of glycosylated integrin β3 (elution) after WGA-mediated isolation of β-1,4-GlcNAc- and sialic acid-linked proteins from total lysates of 4T1 cells upon Phgdh knockdown (4T1 shPHGDH), Cmas knockout, and double Phgdh and Cmas gene inactivation, compared to control cells (4T1 shSCR). Total levels of integrin β3 from the whole cell lysate and actin as loading control are shown. Experiments were performed in triplicate, and one representative experiment is shown. i. β-1,4-GlcNAc- and sialic acid-linked proteins isolated from whole cell lysate of 4T1 cells upon Phgdh and Cmas knockdown (shPHGDH, shCMAS), Cmas knockout (KO) or control (shSCR) cells treated with tunicamycin (0.05 μg ml−1) or DMSO for 72 h (n = 2 independent experiments). Error bars represent s.d. from mean.
Extended Data Fig. 7 Low PHGDH protein expression promotes invasion and metastasis via protein sialylation.
a. Invasive capacity of 4T1 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells pretreated with tunicamycin (0.05 μg ml−1) for 72 h prior seeding in a 3D matrix. The invasive area was stained with calcein green. Each dot represents a different microscopy field (n = 5). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison. b–e. Invasive ability of 4T1 cells with functional (CTR or shSCR) or inactive (Cmas KO or shCMAS) sialic acid pathway upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells(b, d); EMT6.5 cells upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells after 48h of pretreatment with the sialytransferase inhibitor Lith-O-Asp (30 μM) (c); and 4T1 and EMT6.5 cells upon CMAS overexpression (CMAS OE) compared to control (CTR) cells (e) in a 3D matrix. The invasive area was stained with calcein green. Representative images are depicted in the left panel (scale bar 500 μm), quantification in the right panel. Each dot represents a different microscopy field (n = 5). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the smallest and the largest values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison. f. Number of lung metastases per mouse in the orthotopically injected (m.f.p.) mice with 4T1 cells with either functional (CTR, n = 12) or inactive (Cmas KO, n = 10) sialic acid pathway, with (shPHGDH, n = 10) or without (shSCR, n = 10) concomitant Phgdh knockdown. The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the smallest and the largest values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison
Extended Data Fig. 8 Loss of PHGDH protein decreases PKF activity and increases carbon flux into the sialic acid pathway.
a. Changes in metabolite abundance upon Phgdh knockdown in 4T1 and EMT6.5 cells. Data represent fold changes compared to non-silenced cells (shSCR) (n = 3). H6P, hexose-6-phosphate; FBP, fructose bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; xPG, 2/3-phosphoglycerate; PEP, phosphoenolpyruvate. Unpaired t test two-tailed. *indicates statistically significant as follows: FBP, p = 0.0042 (4T1), p = 0.0012 (EMT6.5); PEP, p = 0.0031 (4T1), p = 0.034 (EMT6.5); Pyruvate, p = 2.88e−11 (4T1). b. Fractional contribution of 13C6-glucose to glycolytic intermediates in 4T1 upon Phgdh knockdown (shPHGDH) compared to control (shSCR) cells (n = 3). Error bars represent s.d. from mean. Two-way ANOVA. c. Interaction of PHGDH with PFKP in 4T1 control (shSCR) and Phgdh knockdown (shPHGDH) cells assessed by co-immunoprecipitation of PHGDH. A representative experiment is shown (n = 3 independent experiments). d. Protein expression levels of PHGDH, PSAT and PSPH in TNBC PDX model (BCM-3107-R2TG18) assessed by multiplex immunofluorescence. Green, PHGDH; Turquoise, PSAT; Pink, PSPH; red, pan-cytokeratin tumour marker; blue, DAPI nuclear staining. Scale bar 200 μm. e. Protein expression levels of PHGDH upon treatment with the PHGDH catalytic inhibitor PH-775 (1 μM) for 72 h. One representative experiment is shown (n = 3). f. Inhibition of de novo serine biosynthesis assessed through measurement of serine m+3 labelling after incubation of 4T1 cells for 24 h in culture medium containing 13C6-glucose upon treatment with the PHGDH catalytic inhibitor PH-775 (1 μM) for 72 h. Error bars represent s.d. from mean. Unpaired t test with Welch’s correction, two-tailed. g. Dynamic labelling using 13C6-glucose of 4T1 cells showing 13C incorporation into sialic acid upon treatment with the PHGDH catalytic inhibitor PH-775 (1 μM) for 72h (n = 3). Error bars represent s.d. from mean. Two-way ANOVA. h. Metabolite abundance of sialic acid upon treatment with the PHGDH catalytic inhibitor PH-775 (1 μM) for 72h in 4T1 cells. The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the smallest and the largest values (n = 15). Unpaired t test with Welch’s correction, two-tailed. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; H6P, hexose-6-phosphate; FBP, fructose bisphosphate; DHAP, dihydroxyacetone phosphate; xPG, 2/3-phosphoglycerate; PEP, phosphoenolpyruvate.
Extended Data Fig. 9 Low PHGDH protein expression, but not low catalytic activity, promotes sialic acid synthesis and drives metastatic dissemination.
a, b. Measurement of serine m+3 labelling enrichment after incubation of (a) MDA-MB-231 control (CTR), PHGDH (PHGDH OE) overexpressing and catalytic inactive PHGDH (PHGDH CI OE) overexpressed cells or (b) 4T1 cells upon Phgdh knockdown (shPHGDH), Phgdh-silencing with wildtype (shPHGDH + wt OE) and catalytic inactive overexpression (shPHGDH + CI OE) for 24 h in culture medium containing 13C6-glucose (n = 3). Error bars represent s.d. from mean. One-way ANOVA with Holm-Sidak’s multiple comparison test. c. Dynamic labelling using 13C6-glucose and total abundance of sialic acid in 4T1 cells upon Phgdh knockdown (shPHGDH), Phgdh-silenced cells with wildtype (shPHGDH + wt OE) and catalytic inactive overexpression (shPHGDH + CI OE) cells. Left panel, p values refer to comparison of shSCR or shPHGDH + CI OE vs shPHGDH (n = 3). For sialic acid abundance, the solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values (n = 6). One-way ANOVA with Holm-Sidak’s multiple comparison test. d. Levels of glycosylated integrin β3 (elution) after WGA-mediated isolation of β-1,4-GlcNAc- and sialic acid-linked proteins from total lysates of 4T1 control (shSCR), Phgdh knockdown (shPHGDH) cells, and Phgdh-silenced 4T1 cells with wildtype (shPHGDH +wt OE) and catalytic inactive (shPHGDH + CI OE) overexpression. The last two samples correspond to tunicamycin treated (48 h, 0.05 μg ml−1) 4T1 control (shSCR) and Phgdh knockdown (shPHGDH). Total levels of integrin β3 from the whole cell lysate and actin as housekeeper are shown. One representative experiment is shown (n = 3). e. Invasive capacityof 4T1 cells upon treatment with the PHGDH catalytic inhibitor PH-775 (1 μM) for 72 h and rescue with cell-permeable α-ketoglutarate (αKG, 1 mM) in a 3D matrix. The invasive area was stained with calcein green. Representative images are depicted in the left panel (scale bar 500 μm), quantification in the right panel. Each dot represents a different, randomly selected microscopy field. The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the smallest and the largest values (n = 5). One-way ANOVA with Tukey’s multiple comparison test. f–h. Invasive ability of 4T1 control (shSCR), Phgdh knockdown (shPHGDH) cells and Phgdh-silenced 4T1 cells with wildtype (shPHGDH +wt OE) and catalytic inactive overexpression (shPHGDH + CI OE) (f); MDA-MB-231 control (CTR), PHGDH wildtype overexpression (OE) and catalytic inactive overexpression (CI OE) cells (g); 4T1 control (SCR KO), Psat (PSAT KO) and Psph knockout cells (PSPH KO) (h) in 3D matrix. The invasive area was stained with calcein green. Representative images are depicted in the left panel (scale bar 500 μm), quantification in the right panel. Each dot represents a different microscopy field (n = 5). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and maximum values. Welch and Brown-Forsythe ANOVA with Dunnett’s multiple comparison. i. Percentage of migratory cells per migratory position (n = 11) in the primary tumour of the orthotopic (m.f.p.) 4T1 mouse model assessed by time-lapse intravital imaging. Mice were injected with a mixture of 4T1 shPHGDH-Dendra and 4T1 shPHGDH with overexpression of wildtype (shPHGDH + wt OE) (n = 8) or catalytic inactive (shPHGDH + wt CI) PHGDH-mTurquoise (n = 8). The solid lines indicate the median, the boxes extend to the 25th and 75th percentiles, the whiskers span the minimum and the maximum values. Unpaired t test with Welch’s correction, two-tailed
Extended Data Fig. 10 Validation of genetic modifications of breast cancer cells.
a. Protein expression levels of PHGDH and CMAS in 4T1 cells used in Figs. 2e, 3a–d, and Extended Data Figs. 3j, l, 4a–e, h, 5a, b, e, 6b, e–i, 7a, b, d, f, 8b, c and 9e. b. Protein expression levels of PHGDH and CMAS in 4T1 and EMT6.5 cells used in Extended Data Figs. 2j, 4c. c. Protein expression levels of PHGDH in MDA-MB-231 and 4T1 cells used in Fig. 4c, c and Extended Data Figs. 4f, j, 6c, d, 9a–f and i. d. Relative Phgdh expression in MDA-MB-231 and 4T1 cells detailed in c (n = 3 independent samples). Error bars represent standard deviation (s.d.) from mean. e. Psat and Psph gene inactivation measured by a decrease in 13C6-Glucose incorporation into serine in 4T1 cells used in Extended Data Fig. 9g (n = 3 independent samples). Error bars represent standard deviation (s.d.) from mean. f. Relative Phgdh expression and invasion ability in 4T1 cells used in Figs. 2a, b, 4c, and Extended Data Figs. 3h, i and 9i (n = 3 independent samples). Error bars represent standard deviation (s.d.) from mean.
Supplementary information
Supplementary Figures
Supplementary Figs. 1–3. Supplementary Fig. 1: raw images of the western blot experiments presented in Figs. 3 and 4 and Extended Data Figs. 2, 4, 6 and 8–10. Pictures of exposed uncropped blots are shown together with information on the molecular mass ladder that was used. Supplementary Fig. 2: H&E staining images of adjacent areas of cropped images presented in Fig. 1 and Extended Data Figs. 3, 4 and 8. Supplementary Fig. 3: DNA staining of the adjacent area of the cropped image presented in Extended Data Fig. 1l.
Supplementary Table 1
This table is complementary and related to Fig. 1a, b and Extended Data Fig. 1a–f. It contains clinicopathological information of 129 patients with TNBC.
Supplementary Table 2
This table is complementary and related to Fig. 1d and Extended Data Fig. 2e. It contains clinicopathological information and the location of lymph node metastases of patients with TNBC.
Supplementary Table 3
This table contains the weight of breast primary tumours from mice injected with 4T1 or EMT6.5 and analysed for number of lung metastases (Figs. 2c, 3d and 4d and Extended Data Fig. 7f).
Supplementary Table 4
This table is complementary and related to Fig. 3a. It contains information on metabolites analysed in 4T1 cultured cells including metabolite name, mass isotopomer normalized ion counts and fractional abundances, and total ion counts.
Supplementary Table 5
This table is complementary and related to Extended Data Figs. 6b–d and 9a. It contains information on metabolites analysed in MDA-MB-231 cultured cells, including metabolite name, mass isotopomer normalized ion counts and fractional abundances, and total ion counts.
Supplementary Table 6
This table is complementary and related to Extended Data Fig. 8a. It contains information on metabolites analysed in 4T1 and EMT6.5 cultured cells, including metabolite name, total ion counts and fractional abundances, and the normalization based on protein content measured by BCA.
Supplementary Table 7
This table is complementary and related to Extended Data Fig. 8b. It contains information on metabolites analysed in 4T1 cultured cells including metabolite name, mass isotopomer normalized ion counts and fractional abundances.
Supplementary Table 8
This table is complementary and related to Extended Data Fig. 8e. It contains information on metabolites analysed in 4T1 cultured cells treated with the PHGDH inhibitor PH-755, including metabolite name, mass isotopomer normalized ion counts and fractional abundances.
Supplementary Table 9
This table is complementary and related to Extended Data Fig. 9b-c. It contains information on metabolites analysed in 4T1 cultured cells, including metabolite name, mass isotopomer normalized ion counts and fractional abundances.
Supplementary Table 10
This table contains methods information related to primer sequences.
Supplementary Table 11
This table contains a custom-made gene set related to OPN signalling via AP-1 signature, which was used for GSEA in Fig. 2d and Extended Data Fig. 5a, b.
Supplementary Table 12
This table contains methods information relating to antibodies used for immunofluorescence, fluorescent IHC and IMC.
Supplementary Video 1
Intravital images of 4T1 breast primary tumours generated from a mix of control 4T1 cells (expressing mTurquoise) and Phgdh-silenced cells (expressing Dendra). Representative tracks are presented using solid green (Phgdh-silenced cells) and purple (shControl cells) lines.
Supplementary Video 2
Intravital images of 4T1 breast primary tumours generated from a mix of control 4T1 cells (expressing mTurquoise) and Phgdh-silenced cells (expressing Dendra). Representative tracks are presented using solid green (Phgdh-silenced cells) and purple (shControl cells) lines.
Supplementary Video 3
Intravital images of 4T1 breast primary tumours generated from a mix of Phgdh-silenced cells (expressing Dendra) and Phgdh-silenced cells overexpressing wild-type (video 3) or catalytically inactive (video 4) Phgdh (expressing mCerulean). Representative tracks are presented using solid green (Phgdh-silenced cells) and purple (Phgdh-silenced cells overexpressing wild-type or catalytically inactive Phgdh cells) lines.
Supplementary Video 4
Intravital images of 4T1 breast primary tumours generated from a mix of Phgdh-silenced cells (expressing Dendra) and Phgdh-silenced cells overexpressing wild-type (video 3) or catalytically inactive (video 4) Phgdh (expressing mCerulean). Representative tracks are presented using solid green (Phgdh-silenced cells) and purple (Phgdh-silenced cells overexpressing wildtype or catalytic inactive Phgdh cells) lines.
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Rossi, M., Altea-Manzano, P., Demicco, M. et al. PHGDH heterogeneity potentiates cancer cell dissemination and metastasis. Nature 605, 747–753 (2022). https://doi.org/10.1038/s41586-022-04758-2
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DOI: https://doi.org/10.1038/s41586-022-04758-2
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