Interacting post-muscarinic receptor signaling pathways potentiate matrix metalloproteinase-1 expression and invasion of human colon cancer cells

M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time-and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFRindependent and blocked by inhibiting protein kinase C-alpha (PKC-alpha). Inhibiting activation of PKC- a, EGFR, ERK1/2, or p38-alpha/beta alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-alpha/beta inhibitor. Activating PKC- a and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-alpha/beta and Src, indicating that Src mediates the crosstalk between PKC- a and EGFR signaling. Using siRNA knockdown, we identified p38-alpha as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.