Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: Lactate promotes trastuzumab resistance in gastric cancer by stabilizing HER2 expression. (a) Volcano plot illustrating metabolic differences between gastric cancer tissues from patients who subsequently developed trastuzumab resistance ( n = 6) and those who remained trastuzumab‐sensitive ( n = 6), as determined by untargeted metabolomics analysis. All tissues were surgically resected prior to trastuzumab treatment, and sensitivity or resistance to trastuzumab was subsequently determined based on clinical pathology data and patient follow‐up information. Red dots represent metabolites involved in the glycolytic pathway. (b) Lactate levels in gastric cancer specimens measured using a colorimetric assay kit (all samples assayed in duplicate). Each patient sample is shown in a distinct color, and the two technical replicates from the same patient are shown in the same color. (c) HER2 protein levels measured by enzyme‐linked immunosorbent assay (ELISA) in gastric cancer samples (LAC low , n = 6; LAC High , n = 6). (d,e) HER2 protein levels in AGS cells following treatment with sodium lactate (NALA, 25 or 50 m m ) and lactate (10 or 20 m m ). (f,g) HER2 protein levels in AGS cells following treatment with sodium oxamate (10 or 20 m m ) and stiripentol (100 or 200 µ m ). (h,i) HER2 protein levels in NCI‐N87 cells following treatment with NALA (25 or 50 m m ) and lactate (10 or 20 m m ). (j,k) HER2 protein levels in NCI‐N87 cells following treatment with sodium oxamate (10 or 20 m m ) and stiripentol (100 or 200 µ m ). (l) Flow cytometric analysis of HER2 protein expression in AGS cells treated with NALA or lactate dehydrogenase inhibitors (LDHi) ( n = 3). (m) Flow cytometric analysis of HER2 protein expression in NCI‐N87 cells treated with NALA or LDHi ( n = 3). (n) Representative immunofluorescence (IF) staining and co‐localization analysis of pan‐lactylation and HER2 in gastric cancer tissues (scale bars, 20 µm). (o) Correlation between pan‐lactylation and HER2 expression in gastric cancer tissues (Spearman correlation analysis). (p,q) Lactylation levels of exogenous and endogenous HER2 in cells treated with NALA or LDHi. (r,s) HER2 protein levels in 293T and NCI‐N87 cells treated with LDHi and/or MG132 (10 µ m ). (t,u) Ubiquitination of exogenous and endogenous HER2 following NALA treatment. (v,w) Ubiquitination of exogenous and endogenous HER2 following LDHi treatment. p ‐values were determined by Student's t ‐test; ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Expressing, Analysis, Sensitivity, Pathology, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: AARS1‐mediated HER2 K1171 lactylation enhances its protein stability. (a) Identification of candidate proteins for HER2 lactylation by liquid chromatography‐mass spectrometry (LC‐MS/MS). (b) Screening for the “writer” of HER2 lactylation using co‐immunoprecipitation (Co‐IP). (c) Exogenous Co‐IP assay confirming the interaction between HER2 and AARS1 in 293T cells. (d) Endogenous Co‐IP assays confirming the interaction in NCI‐N87 cells. (e) In vitro lactylation assay showing that AARS1 directly mediates HER2 lactylation. (f,g) IF staining showing the co‐localization of HER2 and AARS1 in human gastric cancer tissue (scale bars, 50 µm) (f) and NCI‐N87 cells (scale bars, 5 µm) (g). (h) Integrating the lactylation sites identified by LC‐MS/MS with DeepKla predictions. (i) Identification of the lactylation site in HER2. (j) Conservation analysis of the lactylation site in HER2 orthologs across species. (k) Western blot analysis of HER2 lactylation in NCI‐N87 cells treated with NALA or LDHi. (l–n) HER2 K1171 lactylation levels following AARS1 overexpression (l), AARS1 knockdown (m), and L‐alanine treatment (n). (o) Lactylation levels of HER2 wild‐type (WT) and the K1171R mutant following AARS1 overexpression. (p,q) Protein levels of HER2 WT and HER2 K1171R mutant following NALA or LDHi treatment. (r–t) Protein levels of HER2 WT and HER2 K1171R mutant following AARS1 overexpression (r), AARS1 knockdown (s), and L‐alanine treatment (t). (u) Western blot analysis of ubiquitination levels in HER2 and the K1171 mutant. (v–x) Ubiquitination of HER2 WT and HER2 K1171R mutant following AARS1 overexpression (v), AARS1 knockdown (w), and L‐alanine treatment (x).

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Stability, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Screening, Co-Immunoprecipitation Assay, In Vitro, Staining, Tissue, Analysis, Western Blot, Over Expression, Knockdown, Mutagenesis, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: K1171 lactylation of HER2 promotes its interaction with USP11. (a,b) HER2 protein levels in 293T and NCI‐N87 cells treated with NALA and/or PR619 (10 µ m ). (c) Deubiquitinases (DUBs) interacting with HER2 identified by LC‐MS/MS analysis and ranked by enrichment score. (d) Knockout of the nine candidate DUBs to identify the key regulator of HER2 protein stability. (e) Western blot analysis of HER2 protein levels in NCI‐N87 cells following USP11 knockout. (f) Western blot analysis of HER2 protein levels in NCI‐N87 cells following USP11 overexpression. (g) HER2 protein levels following overexpression of USP11 WT and its inactive mutant. (h) Ubiquitination levels of HER2 in 293T cells following USP11 overexpression. (i) Ubiquitination levels of HER2 following overexpression of USP11 WT and its inactive mutant. (j,k) Exogenous Co‐IP assays confirming the interaction between HER2 and USP11 in 293T cells. (l) Endogenous Co‐IP assay confirming the interaction between HER2 and USP11 in NCI‐N87 cells. (m) IF staining showing the co‐localization of HER2 and USP11 in NCI‐N87 cells (scale bars, 5 µm). (n,o) Co‐IP analysis of the interaction between MYC‐HER2 and HA‐USP11 following NALA (n) and LDHi (o) treatment. (p–r) Co‐IP analysis of the interaction between HER2 and USP11 binding following AARS1 overexpression (p), AARS1 knockdown (q), and L‐alanine treatment (r). (s) Co‐IP analysis of USP11 binding to HER2 WT and the K1171R mutant. (t) Protein levels of HER2 WT and HER2 K1171R mutant following USP11 overexpression.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Analysis, Knock-Out, Stability, Western Blot, Over Expression, Mutagenesis, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Staining, Binding Assay, Knockdown

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: AARS1‐mediated HER2 lactylation promotes its interaction with EGFR. (a,b) The interaction between exogenously expressed HER2 and EGFR in 293T cells treated with NALA (a) and LDHi (b). (c–e) Co‐IP analysis of endogenous HER2‐EGFR binding in NCI‐N87 cells following NALA (c), LDHi (d), and L‐alanine treatment (e). (f) In vitro GST pull‐down assay showing the effect of AARS1‐induced HER2 lactylation on HER2‐EGFR binding. Coomassie blue staining of purified AARS1 used in the pull‐down assay. (g) Representative IF images and quantitative analysis of HER2 and EGFR in NCI‐N87 cells ( n = 3) following NALA and LDHi treatment (scale bars, 5 µm). (h) Co‐IP analysis of EGFR binding to HER2 WT and HER2 K1171 mutant following NALA treatment. (i) Co‐IP analysis of EGFR binding to HER2 WT and HER2 K1171 mutant following AARS1 overexpression. (j) Representative IF images and quantitative analysis of HER2 K1171 lactylation and EGFR in NCI‐N87 cells ( n = 3) following NALA and LDHi treatment (scale bars, 5 µm). p ‐values were determined by Student's t ‐test; ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Co-Immunoprecipitation Assay, Analysis, Binding Assay, In Vitro, GST Pulldown Assay, Staining, Purification, Pull Down Assay, Mutagenesis, Over Expression

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: Lactate suppression improves trastuzumab efficacy in gastric cancer. (a) Schematic of luciferase‐labeled NCI‐N87 xenograft mouse model establishment. (b) Representative in vivo bioluminescence images of luciferase‐labeled gastric cancer xenografts ( n = 5 per group). All images were acquired under identical exposure parameters and displayed on the same color scale (10,000–60,000). (c) Quantification of tumor burden by region of interest (ROI) analysis over the observation period. (d) Bioluminescence intensity of individual tumors at the end of treatment ( n = 5). (e,f) Flow cytometric analysis and quantification of HER2 expression in mouse tumor tissues ( n = 5). (g,h) Flow cytometric analysis and quantification of EdU incorporation in mouse tumor tissues ( n = 5). (i,j) Representative immunohistochemical (IHC) staining images (scale bars, 50 µm) and quantitative analysis of HER2‐K1171la in mouse tumor tissues ( n = 5, four random fields per tissue section were analyzed). (k,l) Tumor growth curves and Kaplan‐Meier survival analysis of all treatment groups in the NCI‐N87 gastric cancer xenograft model ( n = 6; log‐rank test). (m) Representative images of dissected tumors from the HER2‐overexpressing AGS xenograft model. (n) Tumor volume monitoring in different treatment groups. (o) Volume measurement of dissected tumor ( n = 6). (p) Weight measurement of dissected tumor ( n = 6). p ‐values were determined by two‐way ANOVA (c and n), mixed‐effects models (k), Student's t ‐test (d, f, h, o, and p), or Mann‐Whitney U test (j); ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Luciferase, Labeling, Xenograft Assay, In Vivo, Image Analysis and Quantification, Analysis, Expressing, EdU Assay, Immunohistochemical staining, Immunohistochemistry, Tissue, MANN-WHITNEY

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: Inhibition of HER2 lactylation overcomes trastuzumab resistance in gastric cancer. (a) Representative images of dissected tumors from the NCI‐N87 xenograft model. (b) Tumor volume monitoring in different treatment groups. (c) End‐point volume of dissected tumors ( n = 6). (d) End‐point weight of dissected tumors ( n = 6). (e) Representative images of dissected tumors from the XELOX‐based combination therapy. (f) Tumor volume monitoring in different treatment groups. (g) End‐point volume of dissected tumors ( n = 6). (h) End‐point weight of dissected tumors ( n = 6). (i,j) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). (k) Schematic illustration of the trastuzumab‐resistant gastric cancer mouse model establishment. (l) Representative images of dissected tumors from the resistant xenograft model. (m) Tumor volume curves in the trastuzumab‐resistant model. (n) End‐point volume in the resistant model ( n = 6). (o) End‐point weight in the resistant model ( n = 6). (p,q) Flow cytometric analysis of HER2 expression in tumors from the resistant model ( n = 6). (r,s) Flow cytometric measurement of EdU incorporation in tumors from the resistant model ( n = 6). (t,u) HER2‐K1171la IHC staining (scale bars, 50 µm) and quantification in the resistant model ( n = 6, four random fields per tissue section were analyzed). XELOX, capecitabine plus oxaliplatin. p ‐values were determined by two‐way ANOVA (b, f, and m), Student's t ‐test (d, g, h, and n), Welch's‐ t test (s) or Mann‐Whitney U test (j, o, q, and u); ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Inhibition, Xenograft Assay, Immunohistochemistry, Tissue, Analysis, Expressing, EdU Assay, MANN-WHITNEY

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: Targeting HER2 K1171 lactylation with a peptide inhibitor enhances trastuzumab sensitivity. (a) Schematic illustration of the designed peptide. Green represents the cell‐penetrating peptide (CPP), and red indicates the site for HER2 lactylation. (b,c) Western blot analysis of HER2 lactylation and protein expression in NCI‐N87 cells treated with the peptides. (d,e) Western blot analysis of HER2 lactylation and protein expression in NCI‐N87 cells treated with the peptide‐1# (K1171‐pe). (f) Co‐IP analysis of the binding between endogenous HER2 and EGFR in NCI‐N87 cells treated with K1171‐pe and control peptide (K1171R‐pe). (g) Experimental schematic of the NCI‐N87 xenograft mouse model. (h) Representative images of dissected tumors from the NCI‐N87 xenograft model. (i) Tumor volume monitoring in different treatment groups. (j) Endpoint volume of dissected tumors ( n = 6). (k) Endpoint weight of dissected tumors ( n = 6). (l) Experimental schematic of the patient‐derived xenograft (PDX) model. (m) Representative images of dissected tumors from the PDX model. (n) Tumor growth curves in the PDX model. (o) End‐point volume in the PDX model ( n = 6). (p) End‐point weight in the PDX model ( n = 6). (q,r) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). (s) Representative images of dissected tumors from the HER2‐low expression model. (t) Tumor growth curves in the HER2‐low expression model. (u) End‐point tumor volume in the HER2‐low expression model ( n = 6). (v) End‐point tumor weight in the HER2‐low expression model ( n = 6). (w,x) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). p ‐values were determined by two‐way ANOVA (I, n, and t), Student's t ‐test (j, k, o, p, u, and v), or Mann‐Whitney U test (h, r, and x); ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Sensitivity, Western Blot, Analysis, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Control, Xenograft Assay, Derivative Assay, Immunohistochemistry, Tissue, MANN-WHITNEY

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: HER2 K1171 lactylation is increased in trastuzumab‐resistant tumors and associated with poor prognosis. (a) Representative IF images and quantitative analysis of HER2 K1171 lactylation in trastuzumab‐sensitive and trastuzumab‐resistant gastric cancer tissues (scale bars, 100 µm; n = 6). (b) Representative IHC staining images of human gastric cancer tissues (scale bars, 50 µm). (c–e) Correlation analyses of HER2 expression with LDHA expression (c), AARS1 expression (d), and USP11 expression (e) in gastric cancer tissues ( n = 31; Spearman's rank correlation analysis). (f–h) Correlation analyses of HER2 lactylation with LDHA expression (f), AARS1 expression (g), and USP11 expression (h) in gastric cancer tissues ( n = 31; Spearman's rank correlation analysis). (i) Multicolor IF staining of HER2, LDHA, AARS1, USP11, and HER2 lactylation in gastric cancer tissue microarrays (scale bars, 200 µm; 50 µm for magnified insets). (j–l) Correlation analyses of HER2 expression with LDHA expression (j), AARS1 expression (k), and USP11 expression (l) in the tissue microarray cohort ( n = 96; Spearman's rank correlation analysis). (m–o) Correlation analyses of HER2 lactylation with LDHA expression (m), AARS1 expression (n), and USP11 expression (o) in the tissue microarray cohort ( n = 96; Spearman's rank correlation analysis). (p) Quantification of LDHA expression in gastric cancer tissues across American Joint Committee on Cancer (AJCC) stages I‐IV ( n = 96). (q) Quantification of HER2 lactylation across AJCC stages I‐IV ( n = 96). (r,s) Kaplan‐Meier survival curves showing that high LDHA expression (r) and high HER2 K1171 lactylation (s) are associated with poor overall survival ( n = 96, 48 patients per group; log‐rank test). High and low expression groups were defined based on the median cutoff values (LDHA score: 37.4612; HER2 K1171 lactylation: 26.9943). No cases were censored. p ‐values were determined by Student's t ‐test (a, d, f, and g) or Mann‐Whitney U test (p and q); ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Analysis, Immunohistochemistry, Expressing, Staining, Tissue, Microarray, MANN-WHITNEY