Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR₂), a G-protein-coupled receptor that potently induces β-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR₂ and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.

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.

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

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.