Abstract
Gastrins, including amidated gastrin17 and glycine‐extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21‐activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real‐time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H‐thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K‐dependent pathways. Activated PAK1 in turn mediated gastrin‐stimulated activation of β‐catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.
Highlights
Colorectal cancer (CRC) arises and progresses as a result of cumulative genetic and epigenetic changes in tumor cells
To determine whether gastrins stimulate p21-activated kinase 1 (PAK1) activity in CRC cells, DLD1 cells were stimulated with Gamide or Ggly for 10 min in the presence or absence of LY294002, a PI3K inhibitor, and the activity of PAK1 was determined by measuring the ratio of the phosphorylated and active form of PAK1 to total PAK1 by Western blot
Both Gamide and Ggly significantly increased the phosphorylation of PAK1 to 150% of control (Fig. 1A), and stimulation by either gastrin was blocked by the PI3K inhibitor, LY294002
Summary
Colorectal cancer (CRC) arises and progresses as a result of cumulative genetic and epigenetic changes in tumor cells. Mutations in the Ras and Wnt/b-catenin signaling pathways occur in 50% and 90% of CRCs (Kolligs et al 2002; Yuen et al 2002), respectively. Constitutive activation of Wnt/b-catenin signaling initiates the growth of benign adenomas, while mutations in KRas, BRaf, and related pathways stimulate adenoma growth and contribute to invasive and other malignant behaviors. Accumulation of nuclear b-catenin, and the consequent formation of a constitutively active complex with the transcription factor T-cell factor 4 (TCF4; Korinek et al 1997; Morin et al 1997), promotes cell growth and drives tumor progression through upregulation of target genes such as c-myc (He et al 1998; van de Wetering et al 2002). The nuclear translocation and activation of b-catenin is suppressed by inhibition of p21-activated kinase 1 (PAK1) in CRC cells (He et al 2012), and PAK1 stimulates CRC cell growth in vitro and in vivo by activation of b-catenin
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