Phosphorylation of vasodilator‐stimulated phosphoprotein Ser239 suppresses filopodia and invadopodia in colon cancer

Abstract

In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

Emergent Concepts from the Intestinal Guanylyl Cyclase C Pathway

“…The number and length of cancer cell invadopodia also significantly decreases after activation of the GCC pathway (Zuzga et al, 2011). Importantly, failure to form protrusive structures forces tumor cells to aggregate into compact colonies devoid of spreading and invading abilities (Lubbe et al, 2009;Zuzga et al, 2011). Together, these observations suggest that the GCC pathway is one of the intrinsic homeostatic systems that maintain the stable epithelial cell polarity, shape and tight junctions, which form the essential mucosal barrier between the intestine and the external environment (Han et al, 2011).…”

“…However away from its primary organ, GCC is a ligand-starved receptor with an intracellular dormant pathway, as normal mucosal cells in intestine are the principal producers of endogenous hormones guanylin and uroguanylin (Forte, 1999). Thus, the loss of GCC ligand expression early in transformation (Birkenkamp-Demtroder et al, 2002;Cohen et al, 1998;Notterman et al, 2001;Steinbrecher et al, 2000) may be part of the exclusive phenotypic mutations conferring a pro-metastatic evolutionary advantage to selected colon cancer clones (Lubbe et al, 2009;Pitari et al, 2007;Zuzga et al, 2011).…”

“…Normally restricted at intestinal epithelial brush borders (Lucas et al, 2000), GCC is ideally positioned to affect those molecular networks and exert spatio-temporal control of actin remodelling. Indeed, ligand-dependent GCC signalling through cGMP appears to act as a suppressor of metastatic cell morphology in intestine (Lubbe et al, 2009;Zuzga et al, 2011). Thus, colon cancer cells assume a rounded shape upon GCC signalling activation, with elimination of F-actin rich filopodia and lamellipodia (Lubbe et al, 2009).…”

“…Thus, colon cancer cells assume a rounded shape upon GCC signalling activation, with elimination of F-actin rich filopodia and lamellipodia (Lubbe et al, 2009). The number and length of cancer cell invadopodia also significantly decreases after activation of the GCC pathway (Zuzga et al, 2011). Importantly, failure to form protrusive structures forces tumor cells to aggregate into compact colonies devoid of spreading and invading abilities (Lubbe et al, 2009;Zuzga et al, 2011).…”

“…Hence, dysregulation of GCC signalling in intestinal tumorigenesis may enable the epithelialmesenchymal transition required for cancer cell dissemination (Lubbe et al, 2009). A key intracellular effector of the GCC pathway that regulates colon cancer cell shape is the vasodilator-stimulated phosphoprotein (VASP) (Zuzga et al, 2011). Ena/VASP family proteins control F-actin geometry supporting cell motility (Krause et al, 2003).…”