Additional file 1 of Glucagon signaling via supraphysiologic GCGR can reduce cell viability without stimulating gluconeogenic gene expression in liver cancer cells
Creators: name: Godfrey, Jason
name: Riscal, Romain
name: Skuli, Nicolas
name: Simon, M. Celeste
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Supplementary Figure 1. Liver cancer cells display hypersensitivity to long- and short-term glucose and lipid withdrawal, potentially explained by low gluconeogenic gene expression. (a) Cell number-based proliferation assays of HCC cell lines cultured in different concentrations of glucose. Data represent a single experiment with 3 biological replicates. (b) Cell number-based proliferation assays of HCC cell lines cultured in different concentrations of lipids (oleic acid). Data represent a single experiment with 3 biological replicates. (c) ATP-based cell proliferation assay of HCC cell lines. Data points represent the average of 6 biological replicates. (d) Mutation status of TP53 and CTNNB1 of HCC cell lines. (e) Simplified schematic of opposing glycolytic (red) and gluconeogenic (blue) pathways. G6PC: glucose-6-phosphatase, HX: hexokinase, G6P: glucose-6-phosphate, FBP1: fructose-1,6-bisphophatase 1, PFK: ATP-dependent 6-phosphofructokinase, F-1,6-BP: fructose-1,6-bisphophatase, PCK1: phosphoenolpyruvate carboxykinase (cytosolic), PK: pyruvate kinase. (f) qPCR mRNA expression of gluconeogenic genes in HCC cell lines compared to Primary Human Hepatocytes (PHH). Data represent a single experiment with 3 biological replicates (3 separate RNA samples). ****: p&lt;0.0001, ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (g) Normalized RNA-seq values for gluconeogenic genes in human HCC compared to normal liver. Data obtained from TCGA. ****: p&lt;0.0001, *: p&lt;0.05, unpaired two-tailed t test. n = 50(normal) and 374(tumor). All error bars: +/- SEM. (h) Kaplan-Meier plot of overall survival probability between low and high expression of gluconeogenic enzymes. Graphs were generated using the website: https://kmplot.com . Supplementary Figure 2. Constitutive GCGR expression in SNU398, but not other liver cancer cell lines, stimulates PKA activity in response to glucagon without inducing gluconeogenic gene expression. (a) Normalized RNA-seq values for glycolytic genes in human HCC compared to normal liver. Data obtained from TCGA. ****: p&lt;0.0001, unpaired two-tailed t test. n = 50(normal) and 374(tumor). (b) qPCR mRNA expression of GCGR in Primary Human Hepatocytes (PHH) compared to immortalized, “normal” hepatocyte cell line THLE3. Data represent a single experiment with 3 biological replicates (3 separate RNA samples). (c) Quantification of cAMP in SNU398 expressing either eGFP or GCGR and treated with 100nM glucagon (100G). Each graph represents an independent experiment with 2 technical replicates. DMSO: dimethylsulfoxide, HOAc: 0.05M acetic acid, 20F: 20uM Forskolin (positive control), veh: vehicle (0.05M acetic acid), 20F: 20uM forskolin (positive control). (d) Long-term protein analysis of downstream effectors of cAMP signaling in SNU398 cells expressing either eGFP or GCGR and treated with 3 different concentrations of glucagon. Black triangles denote increasingly equivalent concentrations of vehicle compared to glucagon (red triangles). (e) qPCR mRNA expression of G6PC in SNU398 cells expressing either eGFP or GCGR and treated with 100nM glucagon (100G). Data represent a single experiment with 3 technical replicates (1 RNA sample). Error bars: +/- SD. veh: vehicle (0.05M acetic acid). (f) qPCR mRNA levels of GCGR in HCC cell lines compared to Primary Human Hepatocytes (PHH). Same pLenti-CMV-GCGR or pLenti-CMV-eGFP lentivirus as SNU398 was used for stable expression. Data represent a single experiment with 2 biological replicates (2 separate RNA samples). (g) Quantification of cAMP in HCC cell lines expressing either eGFP or GCGR and treated with 100nM glucagon (100G). Data points represent a single experiment of 2 technical replicates. veh: vehicle (0.05M acetic acid), 20F: 20uM forskolin (positive control). (h) Protein analysis of downstream effectors of cAMP signaling (PKA substrates) in HCC cell lines expressing GCGR and treated with either vehicle (0.05M acetic acid, (-)) or 100nM glucagon (+). (i) qPCR mRNA levels of GCGR and gluconeogenic enzyme genes in cryopreserved primary human hepatocytes (cPHH) treated with 100nM glucagon (100G). Data represent a single experiment with 2 biological replicates (2 separate RNA samples). (j) qPCR mRNA expression of GCGR in SNU398 cells expressing either eGFP or GCGR and treated with 100nM glucagon (100G). Data represent a single experiment with 3 biological replicates (3 separate RNA samples). Error bars: +/- SEM. veh: vehicle (0.05M acetic acid). ns: not significant, p&gt;0.05, ordinary one-way ANOVA with Tukey’s multiple comparisons test. Supplementary Figure 3. Glucagon stimulation of gluconeogenic genes and treatment with epigenetic inhibitors across multiple liver cancer cell lines. (a) qPCR mRNA expression of GCGR, G6PC, FBP1 and PCK1 in HepG2, PLCPRF5 and Huh7 cells expressing either eGFP or GCGR and treated with 100nM glucagon (100G). Data represent a single experiment with 3 technical replicates (1 RNA sample). Error bars: +/- SD. veh: vehicle (0.05M acetic acid). (b) Protein analysis of EZH2 inhibitor efficacy in HepG2, PLC and Hep3B cells at the indicated drug concentrations, culture conditions, and time. (c) Protein analysis of EZH2 inhibitor efficacy in SNU398 cells at the indicated drug concentrations, culture conditions, and time. (d) Protein analysis of DNA methyltransferase inhibitor Decitabine efficacy in PLC cells compared to Primary Human Hepatocytes (PHH) at the indicated drug concentrations, culture conditions, and time. (e) Protein analysis of HDAC inhibitor efficacy in Hep3B, PLC and HepG2 cells at the indicated drug concentrations, culture conditions, and time. Supplementary Figure 4. Glucagon stimulation of GCGR and FBP1 expression and treatment with epigenetic inhibitors across multiple liver cancer cell lines. (a) qPCR mRNA expression of GCGR in THLE3, Normal liver (n=4), Tumor tissue (n=4), HCC cell lines expressing eGFP, VAGA1 PDX tissue and PDX tissue (n=5) compared to Primary Human Hepatocytes (PHH). Data represent a single experiment with 3 biological replicates (3 separate RNA samples). ****: p&lt;0.0001, ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (b) qPCR mRNA levels of GCGR in M7571 cells treated with glucagon plus combinations of epigenetic drugs. Data represent a single experiment with 3 technical replicates (1 RNA sample). Error bars: +/- SD. (-): no drug, 100G: 100nM glucagon, vehicle: 0.035% of 0.05M acetic acid, EZH2i: 1uM GSK126, HDACi: 10nM LBH589, DNMTi: 5uM Decitabine, triple: 1uM GSK126 + 10nM LBH589 + 5uM Decitabine. (c) qPCR mRNA levels of FBP1 in Hep3B, PLC and HepG2 cells treated with glucagon and/or combinations of epigenetic drugs. Data represent a single experiment with 3 technical replicates (1 RNA sample). Error bars: +/- SD. (-): DMSO, EZH2i: 10uM GSK126 (10G), HDACi: 10nM LBH589 (10L), DNMTi: 1uM Decitabine, triple: 10uM GSK126 + 10nM LBH589 + 1uM Decitabine. (d) ATP-based cell proliferation assay of HCC cell lines treated with combinations of epigenetic drugs. Data points represent the average of 3 biological replicates. Error bars: +/- SD. (-): DMSO, EZH2i: 10uM GSK126, HDACi: 10nM LBH589. Supplementary Figure 5. Epigenetic inhibitors reduce cell viability across multiple HCC cell lines but display high toxicity in vivo. (a) ATP-based cell viability assays performed on HCC cell lines treated with serially diluted (1:3) concentrations of epigenetic inhibitors. Data points represent the average of 6 biological replicates. Error bars: +/- SEM. DMSO used a vehicle control. Note, no maximal drug concentration included greater than 0.2% (1:500) of DMSO. Sorafenib used as a clinically relevant drug comparison. UNC0642 (G9a inhibitor (H3K9 dimethylation)) used as an emerging epigenetic target comparison. (b) Crystal violet assay on SNU398 cells treated with EZH2 and pan-HDAC inhibitors. Each well represents a biological replicate. (c) Tumor volume measurements of SNU398 xenografts in Nu/J mice treated with EZH2 (GSK126) and pan-HDAC (LBH589) inhibitors at the indicated dosage. Treatments were performed at irregular intervals (due to loss/recovery of weight) over the course of the experiment by intraperitoneal injection. vehicle: 20% 2-HP-B-CD (hydroxypropyl-beta-cyclodextrin), pH 4.5. N=5 mice per treatment cohort with 2 tumors per mouse. Data points represent average volume of 10 tumors. Error bars: +/- SEM. (d) Body weight measurements of mice treated with EZH2 (GSK126) and pan-HDAC (LBH589) inhibitors at the indicated dosage in same experiment as (c). Data points represent average weights of 5 mice. Error bars: +/- SEM. Supplementary Figure 6. Glucagon/GCGR only decreases cell viability in SNU398 through an unknown mechanism independent of CREB. (a) Cell proliferation assays on liver cancer cell lines either expressing eGFP or GCGR and treated with 100nM glucagon. Data points represent 3 biological replicates. Error bars: +/- SEM. (b) Crystal violet assay on SNU398 cells either expressing eGFP or GCGR, treated with 100nM glucagon (100G), and transfected with 25nM of a small interfering RNA molecular targeting CREB1 (siCREB). Cells were initially treated with glucagon for 3 days and then transfected with siCREB without any further treatment. siNTC (25nM): non-targeting control, veh: vehicle (0.05M acetic acid). (c) Protein analysis of siRNA efficacy in SNU398 cells either expressing eGFP or GCGR and treated with 100nM glucagon. Samples harvested at the 7-day time point as illustrated in the previous figure panel. siCycloB (25nM): Cyclophilin B (positive control for transfection protocol). (d) Protein assessment of the target efficacy of CREB antagonist, 666-15, in SNU398 cells either expressing eGFP or GCGR and treated with or without 100nM glucagon. (e) PI/Annexin V flow cytometry analysis of SNU398 cells expressing either eGFP or GCGR and treated with either 100nM glucagon, 0.5uM 666-15, or the combination. Data points represent average of 3 biological replicates. Error bars: +/- SEM. 10ug/ml blasticidin used as a positive control. ns: not significant, **: adjusted p=0.0055, ordinary one-way ANOVA with Tukey’s multiple comparisons test.