Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 2. Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 3. ARG1/AGMAT determine metabolic gene expression via arginine (A) Immunoblots of SNU-449 cells upon stable expression of ARG1 and/or AGMAT. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in arginine-restricted medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells. N = 6. (D) Arginine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (E) PCA analysis of RNA-seq data of control and ARG1/AGMAT-expressing SNU-449 cells. (F) Heatmap of a subset of differentially expressed metabolic genes in ARG1/AGMAT-expressing compared to control SNU-449 cells (log2 fold-change). (G) mRNA levels of ASNS, PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ARG1/AGMAT-expressing SNU-449 cells. N = 5–7. (H) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (I) Immunoblots of ASNS, PSAT, PSPH, and NNMT of Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) and unpaired t test (G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Gene Expression, Western Blot, Expressing, Control, Growth Assay, RNA Sequencing

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 4. ASNS promotes arginine uptake in liver cancer (A) Relative 3H-arginine uptake in control and ARG1/AGMAT-expressing SNU-449 cells with or without pre-loading with asparagine (Asn) or glutamine (Gln). N = 5–6. (B) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable expression of ASNS or control. Calnexin serves as loading control. (C) Relative 3H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-ex- pressing cells. N = 5. (D) Representative clonogenic growth assay of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells grown in arginine- restricted medium. (E) mRNA levels of PSAT1, PSPH, GLSK, GLUT3, HK2, NNMT, and AOC3 in control and ASNS- expressing SNU-449 ARG1/AGMAT-expressing cells. N = 6–8. (F) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ASNS-expressing SNU-449 ARG1/ AGMAT-expressing cells. Calnexin serves as loading control. (G) mRNA levels of Asns in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV- shCtrl or AAV-shAsns. n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV- shAsns. n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-shAsns. n = 4–6. n.s. = not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (A, C, E, G, and H) and one-way ANOVA (I).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Control, Expressing, Western Blot, Growth Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 7. ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log2 fold-change as indicated. ‘‘?’’ indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T.

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Microarray

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-AGMAT , Novus Biological , Cat#1-82080.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing, Control, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log 2 fold-change as indicated. “?” indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T. (E) Kaplan-Meier survival estimate curve for The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) patients ranked by expression of ARG1 and AGMAT . n = 89 (low), n = 109 (normal). (F) Urea cycle metabolites in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (G) Immunoblots of RBM39 in tumor lysate (Input) and elution after purification with leucine (Leu)- or arginine (Arg)-coupled agarose beads from three HCC patients. Calnexin serves as input and negative control. (H) Dose-response curve of 20 HCC patient-derived organoids treated with indisulam. Data are presented as the percentage of control DMSO-treated tumor organoids. (I) Model. In liver cancer cells, loss of ARG1 and AGMAT preserves arginine, which in turn binds RBM39 to promote metabolic reprogramming. Arginine-RBM39-mediated ASNS expression further enhances arginine uptake. Trsx, transcription. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 by unpaired t test (C), log rank test (E), and multiple t test (F).

Article Snippet: Rabbit anti-AGMAT (for IHC) , Sigma , Cat#PA5-55311.

Techniques: Western Blot, Microarray, Expressing, Purification, Negative Control, Derivative Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1 and AGMAT are decreased and arginine, RBM39, and ASNS are increased in HCC patient tumors that are sensitive to RBM39 depletion by indisulam, related to Figure 7 (A) RBM39 mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (B) RBM39 protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 30 (Edm. low), n = 21 (Edm. high). (C) ASNS mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (D) ASNS protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio, if applicable. BW, black-and-white, i.e., only detected in tumor tissues. n = 3 (Edm. low), n = 8 (Edm. high). (E) Staging of ARG1 and AGMAT IHC staining in tissue micro array. (F) mRNA expression of ARG1 , AGMAT , RBM39 , and ASNS in early-stage HCC (data from Jiang et al. ). log 2 fold-change tumor (T) relative to non-tumor (NT) tissues. n = 35. (G) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of ARG1 . n = 135 (low), n =155 (normal). (H) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of AGMAT . n = 136 (low), n = 158 (normal). (I) Polyamine species in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (J) Arginine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (K) Total polyamine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (L) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of 600 proteins identified by MS (in minimum 2 out of 3 samples) after purification from HCC tissues by arginine (Arg)- compared to leucine (Leu)-coupled agarose beads. Red dot highlights RBM39. (M) Dose-response curve of 20 HCC patient-derived organoids treated with sorafenib. Data are presented as the percentage of control DMSO-treated tumor organoids. (N) IC 50 of indisulam- and sorafenib-treated HCC patient-derived organoids. n = 20. (O and P) Rbm39 and Asns mRNA levels in embryonic day 14 (E14), E18, and adult mouse liver as reads per kilobase of exon per million reads mapped (RPKM). Data from NBCI Gene. n.s. = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by paired t test (A–C, J, K, and N), multiple t test (F and I), and log rank test (G and H).

Article Snippet: Rabbit anti-AGMAT (for IHC) , Sigma , Cat#PA5-55311.

Techniques: Immunohistochemistry, Microarray, Expressing, Purification, Derivative Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet:

Article Snippet: Rabbit anti-AGMAT (for IHC) , Sigma , Cat#PA5-55311.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing Assay, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software