The orexin receptor OX(1)R in colon cancer: a promising therapeutic target and a new paradigm in G protein-coupled receptor signalling through ITIMs

Abstract

An exciting aspect of the heptahelical orexin receptor 1 (OX(1)R) has emerged recently, when it was shown that it drives apoptosis in human colon cancer cell lines. Here we review recent findings related to the role of OX(1)R in colorectal cancers and the unexpected mechanism whereby this G protein-coupled receptor works. The OX(1)R is aberrantly expressed at all steps of primary colorectal tumour progression and after local (lymph node) or distant (liver, lung) metastasis. No OX(1)R is detected in normal colonic epithelial cells. Treatment of human colon cancer cells in culture with orexins promotes robust apoptosis and subsequent reduction of growth including in cells that are resistant to 5-fluorouracil, the most commonly used drug in chemotherapy. When human colon cancer cells are xenografted in nude mice, treatment with orexins dramatically slows tumour growth and even reverses the development of established tumours. Thus, OX(1)R agonists might be novel candidates for colon cancer therapy. Activation of OX(1)R drives apoptosis through G(q) protein but independently of classical Gα(q) activation of phospholipase C. In fact, it is the freed βγ dimer of G(q) that plays a pivotal role by stimulating Src-tyrosine kinase. This results in phosphorylation of two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in OX(1)R and subsequent recruitment by OX(1)R of the phosphotyrosine phosphatase SHP-2, which is activated thereby. Downstream events include release of cytochrome c from mitochondria and activation of caspase-3 and caspase-7. The role of ITIMs in OX(1)R-driven apoptosis represents a new paradigm of G protein-coupled receptor signalling.

Figure 1

Expression of the orexin receptor OX₁R during colon cancer progression and metastasis. OX₁R (blue circles) is aberrantly expressed in primary tumours and metastases. The expression of OX₁R was detected very early during carcinogenesis and whatever the Dukes' stage of the primary tumours. After metastasis in the colon (lymph node) and in distant organs (liver, lung), OX₁R is still expressed. No OX₁R is present in normal colonic epithelial cells from which cancer cells derive. Adapted with permission from © 2005 Terese Winslow, US Government has certain rights.

Figure 2

Mechanism of OX₁R-driven apoptosis. Activation of OX₁R by orexins promotes the dissociation of G_q protein into α_q and βγ dimers. The classical pathway resulting in phospholipase C activation by α_q is not involved in OX₁R-mediated apoptosis and is shown in grey. Freed βγ dimers stimulate the Src-tyrosine kinase resulting in the phosphorylation of the two ITIMs of OX₁R. The phosphotyrosine phosphatase SHP-2 is then recruited by OX₁R and activated thereby. Activated SHP-2 is mandatory for subsequent cytochrome c-mediated mitochondrial apoptosis. PLC, phospholipase C; Cyt c, cytochrome c; DAG, diacylglycerol; SHP-2, SH2 domain-containing phosphotyrosine phosphatase-2. See text for details.

Figure 3

Proposed multi-step mechanisms of activation of OX₁R to account for the high specificity of the OX₁R-driven apoptosis. In step 1, OX₁R is not activated by orexins and the tyrosine residues of ITIMs are not phosphorylated (resting step). During step 2, binding of orexins to OX₁R leads to the G_q protein dissociation from the receptor to yield a Gα_q-GTP monomer and a Gβγ dimer, which are now free to modulate the activity of other intracellular protein. The Gα_q-GTP monomer activates phospholipase C leading to an increase in intracellular Ca⁺⁺, a process that is not involved in the apoptotic response. In Step 3a, the mechanism that could create selectivity in tyrosine phosphorylation of ITIMs by Src is compartmentalization of the signalling complex in which the Gβγ dimer activates Src at the vicinity of the activated OX₁R. Alternatively or additionally, a speculative Step 3b would lead to another ligand-induced change in receptor conformation allowing exposure of tyrosine in the two ITIMs of OX₁R. This step would be necessary for phosphorylation of ITIMs in the receptor. This scheme including Steps 3a and/or 3b would explain why all other GPCRs that are also able to free Gβγ dimers are not able to phosphorylate ITIMs in OX₁R and to promote apoptosis. After tyrosine phosphorylation of ITIMs, the OX₁R processes to further steps by recruiting SHP-2 resulting in the activation of the tyrosine phosphatase and the apoptotic response (see Figure 2).