While the role of G-protein–coupled receptors (GPCR) in cancer is acknowledged, their underlying signaling pathways are understudied. Protease-activated receptors (PAR), a subgroup of GPCRs, form a family of four members (PAR1–4) centrally involved in epithelial malignancies. PAR4 emerges as a potent oncogene, capable of inducing tumor generation. Here, we demonstrate identification of a pleckstrin-homology (PH)-binding motif within PAR4, critical for colon cancer growth. In addition to PH–Akt/PKB association, other PH-containing signal proteins such as Gab1 and Sos1 also associate with PAR4. Point mutations are in the C-tail of PAR4 PH-binding domain; F347 L and D349A, but not E346A, abrogate these associations. Pc(4–4), a lead backbone cyclic peptide, was selected out of a mini-library, directed toward PAR2&4 PH-binding motifs. It effectively attenuates PAR2&4–Akt/PKB associations; PAR4 instigated Matrigel invasion and migration in vitro and tumor development in vivo. EGFR/erbB is among the most prominent cancer targets. AYPGKF peptide ligand activation of PAR4 induces EGF receptor (EGFR) Tyr-phosphorylation, effectively inhibited by Pc(4–4). The presence of PAR2 and PAR4 in biopsies of aggressive breast and colon cancer tissue specimens is demonstrated. We propose that Pc(4–4) may serve as a powerful drug not only toward PAR-expressing tumors but also for treating EGFR/erbB-expressing tumors in cases of resistance to traditional therapies. Overall, our studies are expected to allocate new targets for cancer therapy. Pc(4–4) may become a promising candidate for future therapeutic cancer treatment.

Post‐translational modification of G‐protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post‐translational regulation of protease‐activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell‐surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2, impacting PAR2‐induced signaling and colon cancer growth. RNF43 co‐associates with PAR2, promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R‐spondin2 in the presence of leucine‐rich repeat‐containing G‐protein‐coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased β‐catenin levels, transcriptional activity, phospho‐LRP6, and anchorage‐independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the ‘spleen‐liver’ colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO‐treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the ‘spleen‐liver’ model of spleen‐inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA ‐Par2 silencing. “Limited‐dilution‐assay” performed in mice in‐vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts β‐catenin signaling and colon cancer growth.

G-protein-coupled receptors (GPCRs) are involved in a wide array of physiological and disease functions, yet knowledge of their role in colon cancer stem cell maintenance is still lacking. In addition, the molecular mechanisms underlying GPCR-induced post-translational signaling regulation are poorly understood. Here, we find that protease-activated receptor 4 (PAR4) unexpectedly acts as a potent oncogene, inducing β-catenin stability and transcriptional activity. Both PAR4 and PAR2 are able to drive the association of methyltransferase EZH2 with β-catenin, culminating in β-catenin methylation. This methylation on a lysine residue at the N-terminal portion of β-catenin suppresses the ubiquitination of β-catenin, thereby promoting PAR-induced β-catenin stability and transcriptional activity. Indeed, EZH2 is found to be directly correlated with high PAR4-driven tumors, and is abundantly expressed in large tumors, whereas very little to almost none is expressed in small tumors. A truncated form of β-catenin, ∆N133β-catenin, devoid of lysine, as well as serine/threonine residues, exhibits low levels of β-catenin and a markedly reduced transcriptional activity following PAR4 activation, in contrast to wt β-catenin. Our study demonstrates the importance of β-catenin lysine methylation in terms of its sustained expression and function. Taken together, we reveal that PAR-induced post-transcriptional regulation of β-catenin is centrally involved in colon cancer.

The essential role of G-protein coupled receptors (GPCRs) in tumor growth is recognized, yet a GPCR based drug in cancer is rare. Understanding the molecular path of a tumor driver gene may lead to the design and development of an effective drug. For example, in members of protease-activated receptor (PAR) family (e.g., PAR1 and PAR2), a novel PH-binding motif is allocated as critical for tumor growth. Animal models have indicated the generation of large tumors in the presence of PAR1 or PAR2 oncogenes. These tumors showed effective inhibition when the PH-binding motif was either modified or were inhibited by a specific inhibitor targeted to the PH-binding motif. In the second part of the review we discuss several aspects of some cardinal GPCRs in tumor angiogenesis.

Supplementary Table from PH-Binding Motif in PAR₄ Oncogene: From Molecular Mechanism to Drug Design