Additional file 1: Figure S1a. of Gastrin activates autophagy and increases migration and survival of gastric adenocarcinoma cells

Expression of CCKBR in gastric adenocarcinoma cells. AGS cells have low abundance of the CCKBR, MKN45 express the CCKBR endogenously and AGS-Gr cells are stably transfected with the CCKBR. S1b. Negative control images of the MKN45 cells stained for the CCKBR (phase contrast, Alexa 488, Draq5). Figure S2: Gastrin induces autophagy. AGS-Gr (a & b) cells treated with gastrin (10 nM), BafA1 (100 nM) and gastrin + BafA1 for 2 and 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was analyzed by immunoblotting. The images shown represent one of three independent experiments. Graphs show mean +/- SEM (P- values: *** ≤ 0.01 **≤ 0.02, and * ≤ 0.05). Figure S3: Negative controls (primary antibodies omitted) for MAP1LC3B (Alexa 488) and SQSTM1 (Alexa 647). Figure S4: Gastrin mediated survival is dependent on autophagy. (a): A representative cytometric plots showing AGS-Gr cells treated with BafA1 and gastrin for 18 h. Cell viability was assessed using annexin V-PI staining and flow cytometric analyses. Blocking autophagy reduces gastrin mediated survival in AGS-Gr cells. (b): Cell viability assessed in AGS-Gr cells treated with gastrin (10 nM) for 6- 72 h. (c & d): Cells treated with gastrin (2 h) and subsequently treated with increasing concentrations of cisplatin. Viability was assessed at 24 and 72 h. Results show mean +/-SD (n=3, P-values: * ≤ 0.05 ** ≤ 0.01 *** ≤ 0.001). (e): AGS-Gr cells treated with HCQ for 8 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. (f) Gastrin induced autophagy is dependent on ULK1: AGS-Gr cells treated with gastrin, BafA1 and ULK1 inhibitor SBI-0206965 (10 μM) for 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. The immunoblots represent one of three independent experiments. Figure S5: Inhibition of gastrin induced autophagy by Comp C. AGS-Gr cells pretreated with Compound C (10 μM) for 12 h before adding BafA1 and gastrin (4 h). Protein expression of SQSTM1 is shown by immunoblotting. The blot represents one of two independent experiments. (DOCX 2504 kb

SIK1 inhibits migration in AGS-G_R cells via suppression of MMP-9.

<p><b>A–B:</b> AGS-G_R cells (<b>A</b>) and MKN45 cells (<b>B</b>) were treated with gastrin, and phospho-LKB1 (Ser-428) protein levels determined by Western blot. The phospho-LKB1 bands from a representative experiment are shown. <b>C–D:</b> AGS-G_R cells (<b>C</b>) and MKN45 (<b>D</b>) were treated with gastrin, and phospho-SIK1 (Thr-182) protein levels determined by Western blot. The phospho-SIK1 bands from a representative experiment are shown. <b>E:</b> AGS-G_R cells transfected with siSIK1 or siCtr and real-time cell migration monitored (0–24 h). Results show one representative of three independent experiments (mean ±SD of three technical replicates). <b>F:</b> MMP-9 mRNA expression in cells transfected with pSIK1 and treated with gastrin. Results show one representative of three independent experiments, (mean ± SD).</p

ICER represses the level of <i>SIK1</i> mRNA and protein.

<p><b>A:</b> AGS-G_R cells were treated with gastrin and mRNA levels of ICER measured by qRT-PCR. Results shown are mean ± SEM of three independent biological experiments. <b>B:</b> AGS-G_R cells were transfected with ICER I, ICER IIγ or control expression plasmids, treated with gastrin (1 h) and mRNA levels of SIK1 measured by qRT-PCR. Results show one representative of three independent experiments; mean ± SD of three technical replicates. <b>C:</b> AGS-G_R cells were transfected with siRNAs, treated with gastrin and mRNA levels measured by qRT-PCR. Results show one representative of three independent experiments; mean ± SD of three technical replicates. <b>D:</b> SIK1 Western blot in cells transfected with siICER. A representative image is shown and quantified.</p

Gastrin-induced activation of SIK1.

<p><b>A:</b> AR42J cells were treated with gastrin and mRNA levels measured by qRT-PCR. Mean expression level relative to untreated cells is shown. Results show one representative of three independent biological experiments; mean ± SD of three technical replicates. <b>B:</b> SIK1 Western blot of gastrin treated AR42J cells. A representative image is shown and quantified <b>C:</b> AGS-G_R cells were treated with gastrin and mRNA levels measured by qRT-PCR. Mean ± SEM of three independent biological experiments is shown. <b>D:</b> SIK1 Western blot of gastrin treated AGS-G_R cells. A representative image is shown and the SIK1 bands from two independent experiments were quantified; results shown are mean intensities ±SD. <b>E:</b> SIK1 Western blot of gastrin treated MKN45 cells. The SIK1 bands from a representative experiment were quantified. <b>F:</b> Intracellular localization of endogenous CRTC2 protein (Red; CRTC2, blue; Draq-5-stained DNA). G: Intracellular localization of SIK1 protein. AGS-G_R cells transfected with pEGFP-SIK1.</p

The role of SIK1 in gastrin responsive cells.

<p>Gastrin binds to the CCK2 receptor (CCK2R) and activates the LKB1–SIK1 signalling pathway in adenocarcinoma cells. SIK1 mediated phosphorylation of HDAC leads to cytosolic translocation and activation of transcription. In the gastric adenocarcinoma cell line AGS-G_R ectopic SIK1 inhibits migration.</p

Immunostaining of NR4A2 in gastric adenocarcinoma.

<p><b>A-B</b>: NR4A2 immunoreactivity in normal oxyntic mucosa showing strong intensity in scattered single cells (neuroendocrine cells) and weaker staining intensity in the other epithelial cells. <b>C-F</b>: NR4A2 immunoreactivity in gastric adenocarcinomas of intestinal (C-D) and diffuse (E-F) type, showing a general staining in tumor cells with mixed nuclear or cytoplasmic localization and variable intensities. (A, C, E at x200 magnification, with boxes representing B, D and F at x400 magnification).</p

NR4A2 activates NBRE promoter elements.

<p><b>A</b>: Gastrin-induced NBRE-luc activation. Data represent one of two biological replicas. <b>B</b>: The effect of NR4A2 siRNA on gastrin-induced NBRE activation. Data represent mean ± SEM of four biological replicas (** p<0.01, * p=0.1). <b>C-D</b>: Effect of specific inhibitors of PKA (H-89, 10µM), PI3K (LY 294002, 10µM) or PKC (GF 109203x, 3.5µM) on (<b>C</b>) gastrin-induced NR4A2 gene expression and (<b>D</b>) gastrin-induced NBRE activation. Data represent one of three biological replicas; mean ± SD of six technical replicas.</p

NR4A2 suppresses gastrin-induced migration and invasion.

<p><b>A</b>: Real-time cell migration monitored (0-24 h) in AGS-G_R cells transfected with siNR4A2 or siCtr, with or without gastrin treatment (10 nM). Results show one representative of three biological replicas; mean ±SD of three technical replicas. <b>B</b>: Invasion assay with AGS-G_R cells transfected with pCMX-NR4A2 or pCMX (control) was performed in 24-well plates containing 8-µm pore Matrigel-coated inserts (with or without 0.3 nM gastrin). Cells invading the lower surface of the membrane were stained with Reastain Quick-Diff reagents and total numbers of cells in 5 fields per membrane were counted. The mean of three independent experiments is shown.</p

Negative regulation of gastrin-induced NR4A2 expression.

<p><b>A</b>: AGS-G_R cells transfected with NR4A2-luc and ICER expression plasmids or empty vector. Cells were treated with gastrin for 6 h prior to measurement of NR4A2 activity. Data shown represent mean ± SEM of five biological replicas (** p<0.03, * p = 0.06). <b>B</b>: AGS-G_R cells transfected with NBRE-luc and ICER expression plasmid or empty vector and treated with gastrin for 4 h prior to measurement of NBRE activity. Data shown represent mean ± SEM of four biological replicas (** p<0.03). <b>C</b>: AGS-G_R cells were transfected with pZfp36l1 expression plasmid or empty vector and treated with gastrin (5 nM) NR4A2 mRNA expression was measured by qRT-PCR. Data shown represent one of three biological replicas; mean ± SD of three technical replicas is shown. <b>D</b>: Cyclin L1 represents one of three control genes examined.</p

Melphalan-resistant cells are selectively sensitive to inhibitors targeting enzymes in glycolytic- and pentose phosphate pathways as well as complexes in the mitochondrial electron transport chain.

<p>(A), Bar diagram illustrating effect of inhibitors targeting various enzymes in carbohydrate metabolism (2-DG, 1 mM, 6-AN, 1 mM, SO, 20 mM, DCA, 10 mM) when administered in the presence or absence of 2.5 μM melphalan (Mel). (B), DCA treatment selectively mediates increased mitochondrial ROS in the resistant cells. (C), Western analysis of the apoptotic markers PARP1 (upper panels) and caspase-3 (bottom panels) illustrating increased cleavage of both proteins subsequent to co-treatment of the resistant cells with DCA and melphalan. (D), Bar diagram illustrating effect of inhibitors targeting the mitochondrial electron transport chain or ATP synthesis (metformin, 3 mM, antimycin A, 30 μM, oligomycin, 10 μM) and the general pro-oxidant tBHP (20 μM) when administered in the presence or absence of 2.5 μM melphalan. (E), Western analysis of AKT1 and pAKT1 in the sensitive and resistant cells.</p