Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells

Abstract

Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas.

Figure 1. Gastrin-induced activation of SIK1.

A: 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: SIK1 Western blot of gastrin treated AR42J cells. A representative image is shown and quantified C: AGS-G_R cells were treated with gastrin and mRNA levels measured by qRT-PCR. Mean ± SEM of three independent biological experiments is shown. D: 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. E: SIK1 Western blot of gastrin treated MKN45 cells. The SIK1 bands from a representative experiment were quantified. F: 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.

Figure 2. ICER represses the level of SIK1 mRNA and protein.

A: 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: 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. C: 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. D: SIK1 Western blot in cells transfected with siICER. A representative image is shown and quantified.

Figure 3. SIK1 enhances gastrin-induced transcription.

AGS-G_R cells cotransfected with expression plasmids and reporter plasmids and treated with gastrin (4 h). Results show one representative of three independent experiments; mean ± SD of six technical replicates. A: Cells co-transfected with c-fos-luc and pIRES (control) or pSIK1. B: The effect of pSIK1 wt and pSIK1 mutants on c-fos-luc expression. C: Cells co-transfected with CRE-luc and pIRES (control) or pSIK1. D: Cells cotransfected with pSIK1 and SRE-luc, AP1-luc or NF-κB-luc. E–F: AGS-G_R cells (E) or MKN45 cells (F) transfected with pSIK1 or pIRES (control) and treated with gastrin. Phospho-HDAC4/5/7 was determined by Western blot. For both Western blots one representative of at least two independent biological experiments is shown. G: Intracellular localization of endogenous HDAC4/5/7. Upper panel; untreated cells, lower panel; gastrin treated cells (2 h). H: Intracellular localization of HDAC5 Flag. Upper panel; untreated cells, lower panel; gastrin treated cells (2 h).

Figure 4. SIK1 inhibits migration in AGS-G_R cells via suppression of MMP-9.

A–B: AGS-G_R cells (A) and MKN45 cells (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. C–D: AGS-G_R cells (C) and MKN45 (D) 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. E: 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). F: MMP-9 mRNA expression in cells transfected with pSIK1 and treated with gastrin. Results show one representative of three independent experiments, (mean ± SD).

Figure 5. The role of SIK1 in gastrin responsive cells.

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.