UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis

Abstract

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths1. Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival2,3. However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid4. Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial–mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA.

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Fig. 1: UGDH promotes tumour cell migration by stabilizing SNAI1 mRNA.
Fig. 2: UGDH enhances the binding of HuR to SNAI1 mRNA by converting UDP-Glc to UDP-GlcUA.
Fig. 3: UDP-Glc promotes SNAI1 mRNA decay and impairs lung cancer metastasis.
Fig. 4: pUGDH(Y473) is essential for SNAI1 mRNA stability and lung cancer metastasis.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source Data for Figs. 14 and Extended Data Figs. 110 are included in the online version of the paper. Gel source data can be found in Supplementary Fig. 1.

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Acknowledgements

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000 to W.Y.); the National Natural Science Foundation of China (91857120 and 31471324 to W.Y. and 21625302 and 21573217 to G.L.); CAS Interdisciplinary Innovation Team (JCTD-2018-14 to W.Y.); CAS Facility-based Open Research Program and the Thousand Talents Plan-Youth to W.Y. We thank F. Li from D. Gao’s laboratory for his help downloading and analysing the TCGA datasets; H. Cheng for technical advice; and the Genome Tagging Project (GTP) Center and the Core Facilities of SIBCB for technical support.

Reviewer information

Nature thanks Lydia Finley, Jennifer Gunter and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

W.Y. conceived this study. W.Y., X. Wang and R.L. designed the study. X. Wang performed most of the experiments. R.L. constructed plasmids, generated cell lines, performed TCGA analyses and also contributed to figure editing. R.L. and X. Wang performed the IHC experiments. W.Z., X. Wang and H.Y. performed the animal experiments. G.L. designed the molecular dynamics strategy. G.L. and H.C. performed the simulations and data analysis. H.G., X. Wu, H.Z. and P.W. provided the experimental assistance; J.L. provided reagents and conceptual advice; W.Y. and G.L. wrote the manuscript with comments from all authors.

Correspondence to Guohui Li or Weiwei Yang.

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Competing interests

The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 UGDH is necessary for tumour cell migration.

a, A549 cells were transfected with individual siRNAs targeting 111 rate-limiting metabolic enzymes (four siRNAs per gene), or siRNAs targeting SNAI1 as the positive control (pink wells), or non-targeting siRNA (siNT) as the negative control (grey wells). Scratch wound healing assays were performed. Real-time gap distances were measured using the IncuCyte high-throughput screening system for 24 h. Schematic diagram of the screening strategy is shown. b, Representative images of wound healing assays at the indicated time points performed in A549 cells transfected with or without siUGDH are shown. The protein level of UGDH was examined (left). The gap distance was determined and normalized to that of the siNT group immediately after the scratch (right). c, Schematic diagram of the uronic acid pathway, hexosamine pathway and glycolysis. F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; GlcN6P, glucosamine-6-phosphate; Glu, glucose; Lac, lactic acid; UDP-GlcNAC, UDP N-acetylglucosamine. d, e, A549 or H1299 cells were depleted of endogenous UGDH and rescued with or without rUGDH. d, UGDH expression was examined by immunoblotting analyses. e, Transwell migration assays were performed with genetically engineered H1299 cells. f, A549 cells stably expressing shNT or shUGDH were stably infected with the lentivirus containing luciferase. The luciferase activities were determined. g, Luciferase-expressing A549 cells expressing shNT or shUGDH were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Top, representative images of H&E-stained sections in dissected lungs 35 days after inoculation are shown. Bottom, the metastatic nodules were quantified based on the H&E-stained lung sections. Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test). hj, TCGA RNA-sequencing data of patients with lung adenocarcinoma were analysed. h, UGDH transcription was compared between normal and lung adenocarcinoma tissues (two-tailed Student’s t-test). i, A one-way ANOVA was used to analyse the statistical significance among different stages of lung adenocarcinoma tissues. The boxes represent the median and the first and third quartiles, and the whiskers represent the minimum and maximum of all data points. j, Survival durations of 502 patients with lung adenocarcinoma with low (n = 380, blue curve) or high (n = 122, red curve) expression of UGDH were compared (two-tailed log-rank test). b, d, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. b, e, f, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 2 UGDH regulates SNAI1 mRNA stability.

a, A549 or H1299 cells were stably infected with the lentivirus expressing Flag–SLC35D1. Representative images of immunofluorescence staining with anti-Flag and anti-GLUT1 antibodies from three independent experiments are shown. be, A549 or H1299 cells stably expressing shNT or shUGDH were stably infected with or without the lentivirus expressing SLC35D1. b, Protein expression of SLC35D1 and UGDH was examined. ce, Cells were then supplemented with or without UDP-GlcUA (5 mM) for 16 h (A549) or 24 h (H1299). c, Intracellular UDP-GlcUA concentrations were determined. d, Cellular hyaluronic acid concentrations were determined. e, Transwell migration assays were performed in the H1299 cells treated with or without UDP-GlcUA for 24 h. f, A549 cells stably expressing shNT or shUGDH were supplemented with increasing concentrations of hyaluronic acid for 16 h. The concentrations of cellular hyaluronic acid were determined (left). Transwell migration assays were performed in the presence of increasing concentrations of hyaluronic acid for 16 h (right). g, The mRNA levels of the EMT-related genes CDH1, CDH2, SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, VIM, TCF3, TCF4, FOXC2, SIX1, GRHL2 and ELF5 were examined in A549 cells with or without UGDH depletion by real-time PCR analyses. h, The mRNA levels of SNAI1, CDH1 and CDH2 were examined in UGDH-depleted H1299 cells rescued with or without rUGDH by real-time PCR analyses. i, Protein expression of SNAIL, N-cadherin and E-cadherin was examined in UGDH-depleted A549 or H1299 cells rescued with or without rUGDH by immunoblotting analyses. j, UGDH-depleted A549 cells rescued with or without rUGDH were transfected with a SNAI1 promoter–luciferase reporter construct. Luciferase activities were determined. Relative luciferase activities were normalized to the luciferase activities of tumour cells expressing shNT and the luciferase activities of the Renilla control plasmid. k, UGDH-depleted H1299 cells were rescued with or without rUGDH. The mRNA stability of SNAI1 was determined by treating cells with actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined at the indicated time points by real-time PCR analyses. b, i, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. ch, j, k, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 3 UGDH promotes tumour cell migration through SNAIL.

ac, A549 cells stably expressing shNT or shUGDH were stably infected with the lentivirus expressing SLC35D1. Subsequently, the A549 cells were supplemented with or without UDP-GlcUA (5 mM). The mRNA stability of SNAI1 was determined by treating the A549 cells with actinomycin D (1 μg ml−1). a, The remaining SNAI1 mRNA levels were examined at the indicated time points by real-time PCR analyses. b, The mRNA levels of SNAI1 were examined by real-time PCR analyses. c, The protein expressions of SNAIL and UGDH were examined. d, e, A549 or H1299 cells stably expressing shNT or shUGDH were infected with or without the lentivirus expressing SNAIL. d, Protein expression of SNAIL and UGDH was examined by immunoblotting analyses. e, Transwell migration assays were performed. f, A549 or H1299 cells stably expressing shNT or shUGDH were infected with or without the lentivirus expressing shSNAIL and the lentivirus expressing rSNAIL. Protein expression of SNAIL and UGDH was examined. g, A549 or H1299 cells stably expressing shNT or shSNAIL were infected with or without the lentivirus expressing rSNAIL. Protein expression of SNAIL was examined. h, Transwell migration assays were performed in cells generated as described in f. i, A549 cells were treated with or without the EGFR inhibitor afatinib (2 μM) for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. j, A549 cells were pretreated with or without afatinib (1 μM) for 3 h, followed by EGF treatment (100 ng ml−1) for 30 min. k, A549 cells were treated with increasing doses of EGF for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. l, A549 cells (left) or H1299 cells (right) with or without UGDH depletion were treated with or without EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. c, d, f, g, j, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. a, b, e, h, i, k, l, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 4 UGDH interacts with HuR to regulate SNAI1 mRNA stability.

a, Mass spectrometry analyses of UGDH-associated proteins in A549 cells treated with or without EGF (100 ng ml−1) for 30 min. The data are presented as a volcano plot for two biologically independent experiments (two-tailed Student’s t-test). Proteins with P < 0.0001 and log2-transformed fold change in expression of >2.0 were regarded as the candidate proteins that showed strong interactions with UGDH. FC, fold change. b, A549 cells co-transfected with HA–HuR and Flag–UGDH were treated with or without EGF (100 ng ml−1) for 30 min. c, H1299 cells were treated with or without EGF (100 ng ml−1) for 30 min. d, A549 cells were treated with or without EGF (100 ng ml−1) for 30 min. Nuclear/cytosolic fractionation assays were performed. e, A549 cells were treated with or without EGF (100 ng ml−1) for 30 min. Immunofluorescence staining was performed with anti-UGDH and anti-HuR antibodies. Representative images from three independent experiments are shown. f, Immunofluorescence staining was performed in A549 cells stably expressing shNT or shUGDH using an anti-UGDH antibody. Representative images from three independent experiments are shown. g, Immunofluorescence staining was performed in A549 (top), H1299, PC-9, SMMC-7721, SW-480 and DU-145 cells using an anti-UGDH antibody (bottom). Representative images from three independent experiments are shown. h, ELAVL1 was depleted in A549 or H1299 cells. i, H1299 cells with or without ELAVL1 depletion were treated with or without EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. j, k, A549 cells stably expressing shNT or shUGDH were stably infected with or without the lentivirus expressing shHuR. j, Protein expression of UGDH and HuR in A549 cells was examined. k, The mRNA stability of SNAI1 was determined by treating the A549 cells with or without EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. bd, h, j, Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. i, k, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 5 UGDH activity is required for HuR binding to SNAI1 mRNA.

a, A549 cells were transfected with SFB–SNAI1-ORF–3′ UTR(WT) or its deletion mutants, including SFB–SNAI1-ORF–3′ UTR(ΔARE1), SFB–SNAI1-ORF–3′ UTR(ΔARE2) or SFB–SNAI1-ORF–3′ UTR(ΔARE3). Cells were treated with EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). The remaining SFB-SNAI1 mRNA levels were examined by real-time PCR analyses. b, Purified recombinant His–HuR (10 μg) was incubated with biotin–ARE1 (200 nM) for 4 h. Pull-down assays were performed with streptavidin agarose beads. c, H1299 cells with or without UGDH depletion were treated with or without EGF (100 ng ml−1) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without biotin–ARE1 for 4 h. d, A549 or H1299 cells with or without UGDH depletion were treated with or without EGF (100 ng ml−1) for 4 h. RNA immunoprecipitation assays were performed in the cells using anti-HuR antibody, followed by real-time PCR analyses of precipitated SNAI1 mRNA. Relative precipitated RNA levels were normalized to those of the cells expressing shNT without EGF treatment. e, A549 cells were infected with the lentivirus expressing Flag–UGDH(WT) or Flag–UGDH(ED). Flag–UGDH(WT) or Flag–UGDH(ED) was immunoprecipitated using cell lysates for the measurement of enzymatic activity. f, g, UGDH-depleted A549 cells or H1299 cells were rescued with rUGDH(WT) or rUGDH(ED). f, The expression of UGDH was examined by immunoblotting analyses. g, The concentrations of intracellular UDP-Glc and UDP-GlcUA were determined. h, The concentrations of intracellular UDP-Glc and UDP-GlcUA were determined in A549 cells. i, UGDH-depleted H1299 cells were rescued with rUGDH(WT) or rUGDH(ED). The cells were treated with or without EGF (100 ng ml−1) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without biotin–ARE1 for 4 h. j, The complex of HuR with RNA (PDB code 4ED5) is superimposed on the HuR–UDP-Glc and HuR–UDP-GlcUA complexes. The protein and RNA are shown as cartoon and are coloured cyan and green, respectively. UDP-Glc and UDP-GlcUA are shown as sticks. UDP-Glc, UDP-GlcUA and RNA (U6–U10) in the two ligand binding sites are all represented by a van der Waals surface, and the van der Waals surfaces are coloured orange, pink and green, respectively. b, c, f, i, Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. a, d, e, g, h, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 6 UDP-Glc inhibits mRNA binding of HuR and tumour metastasis.

a, Isothermal titration calorimetry assays were performed with His–RRM1/2(WT) (0.05 mM), His–RRM1/2(Y63F) (0.05 mM) or His–RRM1/2(N134A) (0.05 mM) and UDP-Glc (0.5 mM, left) or UDP-GlcUA (0.5 mM, right). Data are representative of three independent experiments. b, Purified recombinant His–RRM1/2(WT) or His–RRM1/2(N134A) (2 μg) was incubated with biotin–ARE1 (0.5 μM) for 4 h, followed by pull-down assays. c, Purified recombinant His–RRM1/2(WT) or His–RRM1/2(N134A) (2 μg) was incubated with biotin–ARE1 (5 μM) for 4 h in the absence or presence of UDP-Glc (25 μM), followed by pull-down assays. d, e, ELAVL1-depleted A549 or H1299 cells were rescued with rHuR(WT) or rHuR(N134A). d, The expression of HuR was examined. e, Cells were treated with or without EGF (100 ng ml−1) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without biotin–ARE1 for 4 h. f, ELAVL1-depleted H1299 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml−1) for 4 h. RNA immunoprecipitation assays were performed using an anti-HuR antibody, followed by real-time PCR analyses of precipitated SNAI1 mRNA. Relative precipitated RNA levels were normalized to those of the cells rescued with rHuR(WT) without EGF treatment. g, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml−1) for 4 h. RNA immunoprecipitation assays were performed using anti-HuR antibody, followed by real-time PCR analyses of precipitated mRNA transcribed by indicated genes. Relative precipitated RNA levels were normalized to those of the cells rescued with rHuR(WT) without EGF treatment. h, i, ELAVL1-depleted H1299 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). h, The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. i, Transwell migration assays were performed with cells treated with or without EGF (100 ng ml−1) for 24 h. j, Cell proliferation of ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) was determined. k, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were stably infected with the lentivirus expressing luciferase. Subsequently, luciferase activities were determined. l, Luciferase-expressing, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Top, representative images of H&E-stained sections in dissected lungs 30 days after inoculation are shown. Bottom, the metastatic nodules were quantified based on the H&E-stained lung sections. Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test. be, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. fk, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 7 UGDH promotes tumour metastasis by relieving the inhibition of HuR by UDP-Glc.

a, Transwell migration assays were performed in A549 cells treated with increasing concentrations of UDP-Glc for 16 h. bd, A549 cells stably expressing luciferase were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Then, 14 days after inoculation, mice were injected with or without UDP-Glc (200 mg per kg, every 2 days) by tail-vein injection. b, Left, 28 or 30 days after inoculation, bioluminescence imaging of the tumour-implanted mice was carried out and representative images of lung metastasis are shown. Right, the luciferase intensities of metastatic tumours in the lung were statistically analysed. The relative luciferase intensities were normalized to those of the mice implanted with A549 cells without UDP-Glc treatment at day 28. Data represent the mean ± s.d. of the luciferase intensities in six mice (two-tailed Student’s t-test). c, Top, representative images of H&E-stained sections in dissected lungs 35 days after inoculation are shown. Bottom, the metastatic nodules were quantified based on the H&E-stained lung sections. Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test). d, Kaplan–Meier survival analysis of a different group of mice (nine mice per group) implanted with tumour cells with or without UDP-Glc administration (two-tailed log-rank test). Upwards tick mark represents censored (alive at last follow-up) mice. e, f, UGP2-depleted A549 or H1299 cells were rescued with or without rUGP2. e, The expression of UGP2 was examined. f, The intracellular UDP-Glc levels were determined. g, h, A549 or H1299 cells stably expressing shNT or shUGDH were infected with or without the lentivirus expressing shUGP2. g, The expression of UGP2 and UGDH was examined. h, Transwell migration assays were performed with cells treated with EGF (100 ng ml−1) for 16 h (A549 cells) or 24 h (H1299 cells). i, j, A549 cells stably expressing shNT or shUGDH were infected with or without the lentivirus expressing shUGP2. Cells were stably infected with the lentivirus expressing luciferase and then implanted into randomized athymic nude mice by tail-vein injection (six mice per group). i, Left, 25 days after inoculation, bioluminescence imaging of tumour-implanted mice was carried out and representative images of lung metastasis are shown. Right, the luciferase intensities of metastatic tumours in lung were statistically analysed. The relative luciferase intensities were normalized to those of the mice implanted with A549 cells expressing shNT. Data represent the mean ± s.d. of the luciferase intensities in six mice. j, The metastatic nodules were quantified based on the H&E-stained lung sections. Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test). k, l, ELAVL1-depleted A549 or H1299 cells rescued with rHuR(WT) or rHuR(N134A) were infected with or without the lentivirus expressing shUGDH. k, The expression of HuR and UGDH was examined. l, Transwell migration assays were performed with cells treated with EGF (100 ng ml−1) for 16 h (A549 cells) or 24 h (H1299 cells). e, g, k, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. a, f, h, l, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 8 UGDH–HuR interaction requires MEK1-dependent pUGDH(Y473).

a, A549 cells were treated with EGF (100 ng ml−1) for 30 min. HuR was immunoprecipitated and treated with or without RNase (10 ng μl−1) for 30 min. b, A549 cells were infected with or without the lentivirus expressing HA–HuR or Flag–UGDH and then treated with EGF (100 ng ml−1) for 30 min. The immunoprecipitated complex was treated with or without calf intestinal alkaline phosphatase (CIP) for 30 min. c, A549 cells stably expressing Flag–UGDH were treated with or without EGF (100 ng ml−1) for 30 min. d, e, Mass spectrometry analyses of UGDH-associated proteins were performed in A549 cells stably expressing Flag–UGDH treated with or without EGF (100 ng ml−1) for 30 min. d, All phosphorylated tyrosine residues are listed. The precursor ion was fragmented by collision-induced dissociation and analysed using an ion trap. The database search engine (Andromeda) score was matched to the identified peptide. e, Mass spectrometry analyses of phosphorylation of Y108 in 103-AADLKpYIEACAR-114, phosphorylation of Y352 in 340-KDTGDTRESSSIpYISK-355, and phosphorylation of Y473 in 471-IPpYAPSGEIPK-481 were performed. The parameters including scan number, method, score and m/z are shown. f, A549 cells stably expressing Flag–UGDH(WT), Flag–UGDH(Y108F), Flag–UGDH(Y352F) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml−1) for 30 min. g, H1299 cells stably expressing Flag–UGDH(WT) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml−1) for 30 min. h, A549 cells (left) or A549 cells stably expressing Flag–UGDH(WT) (right) were treated with or without TGFβ (20 ng ml−1) for 30 min. i, A549 cells stably expressing Flag–UGDH were pretreated with or without the MEK inhibitor U0126 (0.25 μM) for 4 h or the p38 inhibitor BIRB 796 (1 μM) for 4 h, followed by treatment with or without EGF (100 ng ml−1) for 30 min. j, A549 cells were treated with or without the MEK inhibitor U0126 (0.25 μM) for 4 h (top) or the p38 inhibitor BIRB 796 (1 μM) for 4 h (bottom). k, A549 or H1299 cells stably expressing Flag–UGDH were infected with or without the lentivirus expressing shMEK1. The expression of MEK1 in the cells was examined (left). Cells were treated with or without EGF (100 ng ml−1) for 30 min (right). l, A549 cells stably expressing shNT or shUGDH were treated with EGF (100 ng ml−1) for 30 min. m, A549 cells stably expressing Flag–UGDH(WT) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml−1) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without purified recombinant His–RRM1/2. n, Flag–UGDH(WT) or Flag–UGDH(Y473F) were immunoprecipitated to determine UGDH enzymatic activity. Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). ac, fn, Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. Source data

Extended Data Fig. 9 pUGDH(Y473) is required for tumour cell migration but not for proliferation.

a, UGDH-depleted A549 or H1299 cells were rescued with rUGDH(WT) or rUGDH(Y473F). bd, UGDH-depleted H1299 cells rescued with rUGDH(WT) or rUGDH(Y473F) were treated with or without EGF (100 ng ml−1) for 4 h. RNA immunoprecipitation assays were performed using anti-HuR antibody, followed by real-time PCR analyses of precipitated SNAI1 mRNA. b, Relative precipitated RNA levels were normalized to those of the cells rescued with rUGDH(WT) without EGF treatment. c, d, Cells were treated with or without EGF (100 ng ml−1) for the indicated times in the presence of actinomycin D (1 μg ml−1). c, The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. d, Transwell migration assays were performed with cells treated with or without EGF (100 ng ml−1) for 24 h. e, UGDH-depleted A549 cells were rescued with rUGDH(WT) or rUGDH(Y473E) (left). Transwell migration assays were performed with cells treated with or without EGF (100 ng ml−1) for 16 h (right). f, UGDH-depleted A549 or H1299 cells were rescued with rUGDH(WT) or rUGDH(Y473F). The cells rescued with rUGDH(Y473F) were infected with or without the lentivirus expressing SNAIL (left). Transwell migration assays were performed with cells treated with EGF (100 ng ml−1) for 16 h (A549 cells) or 24 h (H1299 cells) (right). g, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were stably infected with the lentivirus expressing luciferase. Subsequently, luciferase activities were determined. h, Luciferase-expressing, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Representative images of H&E-stained sections in dissected lungs 35 days after inoculation are shown (top). The metastatic nodules were quantified based on the H&E-stained lung sections (bottom). Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test). ik, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were stably infected with the lentivirus expressing luciferase and then orthotopically implanted into the lungs of randomized athymic nude mice (six mice per group). i, Left, 15 days after inoculation, bioluminescence imaging of implanted mice was carried out and representative images of lung tumours are shown. Right, the luciferase intensities of lung tumours were statistically analysed. The relative luciferase intensities were normalized to those of the mice implanted with UGDH-depleted A549 cells rescued with rUGDH(WT). Data represent the mean ± s.d. of the luciferase intensities in six mice. j, Top, representative images of dissected lungs and H&E-stained sections in dissected lungs 15 days after inoculation are shown. Bottom, the percentage of tumour area was quantified based on the H&E-stained lung sections. The relative percentage of metastatic tumour area was normalized to that of mice implanted with cells rescued with rUGDH(WT). Data represent the mean ± s.d. of the relative percentage of tumour area per mouse in six mice. k, Left, representative images of IHC staining of tumours with anti-Ki-67 antibody are shown. Right, the relative percentages of Ki-67+ cells were quantified. Data represent the mean ± s.d. of the relative percentage of Ki-67+ cells in six mice (two-tailed Student’s t-test). a, e, f, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. bg, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

Extended Data Fig. 10 pUGDH(Y473) correlates with SNAIL expression and metastatic recurrence.

a, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) (pGIPZ-shUGDH also contains the GFP ORF) were implanted into randomized athymic nude mice by tail-vein injection (five mice per group). Subsequently, 3 h after inoculation, the mice were euthanized. Representative images of intravascular tumour cells (green) out of lung blood vessels (red) are shown. The numbers of tumour cells in lungs (including intracellular and extravascular tumour cells) were quantified. Data represent the mean ± s.d. of the relative number of tumour cells per mouse in five mice normalized to the control group. b, c, Cell proliferation (b) and colony formation (c) were determined in A549 cells stably expressing shNT or shUGDH, or UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F). d, A549 cells stably expressing shNT or shUGDH, or UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were subcutaneously injected into randomized athymic nude mice (six mice per group). The weight of xenograft tumours was statistically analysed. Data represent the mean ± s.d. of relative tumour weight in six mice (two-tailed Student’s t-test). e, Validation of antibody specificities. IHC analyses of human lung cancer specimens were performed with the indicated antibodies in the presence or absence of specific blocking peptides or proteins. Data are representative of three biologically independent experiments. f, g, IHC analyses of 114 specimens from patients with lung cancer were performed using anti-pUGDH(Y473) and anti-SNAIL antibodies. f, Representative images of IHC staining of three lung cancer specimens are shown. g, Semi-quantitative scoring (using a scale from 0 to 300) was carried out, Pearson’s correlation test. h, IHC analyses were performed in primary tumours from patients with lung cancer with or without metastatic recurrence using anti-pUGDH(Y473) antibody. Staining scores of pUGDH(Y473) were compared between the patients with metastatic recurrence (n = 50) and the patients without metastatic recurrence (n = 50). Data represent the mean ± s.d. of the two groups (two-tailed Student’s t-test). i, IHC staining was performed in primary tumours and paired metastatic tumours from 16 patients with lung cancer with distant metastasis using an anti-pUGDH(Y473) antibody. Staining scores of the primary lung cancer specimens were compared with those of metastatic lung cancer specimens. Data represent the mean ± s.d. of the two groups (two-tailed paired Student’s t-test). j, Schematic model of the mechanism of UGDH-regulated tumour cell migration. Upon EGFR activation, phosphorylated UGDH(Y473) interacts with HuR and converts UDP-Glc into UDP-GlcUA, which releases HuR to bind to and stabilize SNAI1 mRNA. Increased SNAIL expression consequently promotes tumour cell migration. Exo, exonuclease. b, c, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). Source data

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