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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.

Cancer mucosa antigens are emerging as a new category of self-antigens expressed normally in immunologically privileged mucosal compartments and universally by their derivative tumors. These antigens leverage the established immunologic partitioning of systemic and mucosal compartments, limiting tolerance opposing systemic antitumor efficacy. An unresolved issue surrounding self-antigens as immunotherapeutic targets is autoimmunity following systemic immunization. In the context of cancer mucosa antigens, immune effectors to self-antigens risk amplifying mucosal inflammatory disease promoting carcinogenesis. Here, we examined the relationship between immunotherapy for systemic colon cancer metastases targeting the intestinal cancer mucosa antigen guanylyl cyclase C (GCC) and its effect on inflammatory bowel disease and carcinogenesis in mice. Immunization with GCC-expressing viral vectors opposed nascent tumor growth in mouse models of pulmonary metastasis, reflecting systemic lineage-specific tolerance characterized by CD8(+), but not CD4(+), T-cell or antibody responses. Responses protecting against systemic metastases spared intestinal epithelium from autoimmunity, and systemic GCC immunity did not amplify chemically induced inflammatory bowel disease. Moreover, GCC immunization failed to promote intestinal carcinogenesis induced by germ-line mutations or chronic inflammation. The established role of CD8(+) T cells in antitumor efficacy, but CD4(+) T cells in autoimmunity, suggests that lineage-specific responses to GCC are particularly advantageous to protect against systemic metastases without mucosal inflammation. These observations support the utility of GCC-targeted immunotherapy in patients at risk for systemic metastases, including those with inflammatory bowel disease, hereditary colorectal cancer syndromes, and sporadic colorectal cancer.