Protease-activated receptor 2 (PAR2): A new target for RNF43 regulation centrally impacting on colon cancer

Significance 

In the combat of most gastrointestinal (GI) cancers, it is yet challenging to define a selective patient population for the best response of therapeutic medicament/s, resulting in mediocre to low response rates especially in patients with advanced disease.  Many GI cancers, especially colorectal cancer (CRC) and pancreatic cancer (PC), exhibit alterations in the Wnt signaling pathway, which could be used as targets for novel therapy modalities. Wnt signaling plays a pivotal role in tissue homeostasis, development, and cancer. It is mediated by frizzled (FZD) receptors belonging to the G-protein coupled receptor (GPCR) family, activated via Wnt ligands for the initiation of  β-catenin signaling pathway.

Colon cancer is a type of cancer that originates in the large intestine, the final part of the digestive tract. It is one of the most common and deadly cancers worldwide, affecting millions of people and causing hundreds of thousands of deaths every year. Colon cancer usually develops from benign growths called polyps that form on the inner lining of the colon. Some of these polyps can undergo malignant transformation and invade the surrounding tissues and organs, or spread to distant sites through the bloodstream or lymphatic system. The molecular mechanisms underlying colon cancer initiation and progression are complex and involve multiple genetic and epigenetic alterations that affect various signaling pathways. One of these pathways is the Wnt/β-catenin signaling pathway. GPCRs are involved in many physiological and pathological processes, including inflammation, metabolism, immunity, and cancer. Protease-activated receptors (PARs) are a subgroup of GPCRs that are activated by proteolytic cleavage of their extracellular domains by different proteases. Among the four members of the PAR family (PAR1-4), PAR2 has been implicated in colon cancer development and progression. Post-translational modifications (PTMs) are chemical modifications that occur on proteins after their synthesis and affect their structure, function, stability, and interactions. One of the most common PTMs of GPCRs is ubiquitination, which involves the covalent attachment of ubiquitin molecules to lysine residues on the target proteins. RNF43 is a cell-surface transmembrane E3 ubiquitin ligase that belongs to the RING finger family. RNF43 acts as a tumor suppressor in several cancers by inhibiting Wnt/β-catenin signaling and limiting stem cell expansion.

Understanding the role of RNF43 in colon cancer can provide insights into potential therapeutic targets and personalized treatment approaches for this disease. A pioneering finding by Hao et al 1 and Koo et al.,2 revealed that E3 ubiquitin ligase RNF43 and ZNRF3 are regulators of FZD receptors. Both act as negative feedback controls of the FZD family via ubiquitin-mediated, lysosomal degradation1-3. R-spondin binding to LGR54,5, another GPCR, releases FZD inhibition, causing the internalization of ZNRF3/RNF43. It consequently rescues FZD degradation leading to the amplification of Wnt signaling1-3,5. In a new research published in the peer-reviewed Journal, The FASEB Journal, researchers Jeetendra Nag, Priyanga Appasamy, Shoshana Sedley, Hodaya Malka, Tatyana Rudina, and led by Professor Rachel Bar- Shavit from the Hadassah-Hebrew University Medical Center hypothesized that RNF43, a cell-surface transmembrane E3 ubiquitin ligase, is a negative feedback regulator of PAR2, a subgroup of GPCRs, and that this regulation affects β-catenin signaling and colon cancer growth6. In-fact, PAR2 competes with FZD as targets of RNF43 activity. Hence a new partner for RNF43 regulation is established, which centrally impacts on PAR2 mediated key β-catenin  pathway in colon cancer.

The research team found that RNF43 co-immunoprecipitates with PAR2 and promotes its ubiquitination and degradation, leading to reduced cell surface expression and attenuated downstream gene signature such as cylin D1 and c-Myc. Researchers also showed that RNF43 suppresses PAR2-induced cell migration and invasion in colon cancer cell lines. However, they observed that R-spondin 2, a ligand for LGR5, rescues PAR2 degradation by RNF43 and enhances its signaling and function. Moreover, they demonstrated that PAR2 and LGR5 act synergistically to activate β-catenin signaling and increase anchorage-independent growth in colon cancer cells. They validated the expression levels of PAR2 and RNF43 in clinical samples and found that PAR2 expression is higher while RNF43 expression is lower in colon cancer tissues. To assess the in vivo role of PAR2 in colon cancer, they used two animal models: a chemically induced colon cancer model by AOM/DSS treatment and a spleen-liver metastasis model. The authors found that PAR2 knockout mice are resistant to colon cancer induction by AOM/DSS treatment compared to wild-type mice, and that RNF43 expression is higher in the colon tissues of PAR2 knockout mice than in those of wild-type mice after AOM/DSS treatment. In elegant molecular and cellular experiments, they also found that PAR2 silencing by shRNA reduces colon cancer cell metastasis to the liver in the spleen-liver model, and that RNF43 expression is higher in the liver tissues of mice with shRNA-Par2 than in those with shRNA-control after spleen injection of colon cancer cells. Additionally, they performed a limited dilution assay to evaluate the cancer stem cell potential of PAR2, and found that PAR2 is enriched in the cancer stem cell population of colon cancer cells.

Taken together, the findings of Professor Rachel Bar-Shavit and colleagues revealed a novel partner for RNF43 regulation impacting on colon cancer growth and development. They uncovered a new important axis of β-catenin signaling modulation by the interaction between PAR2 and LGR5 in colon cancer cells. The findings suggest that targeting PAR2, RNF43, or their interplay could be a potential therapeutic strategy for colon cancer.

Protease-activated receptor 2 (PAR2): A new target for RNF43 regulation centrally impacting on colon cancer - Medicine Innovates
Legend: I. PAR2 induces β-catenin stabilization and β-catenin transcriptional activity leading to downstream gene signature. II. RNF43, an E3 ubiquitin ligase degrades cell surface PAR2, hence inhibits PAR2 induced β-catenin stability and transcriptional activity. III. R-spondin (RSPO) when ligates to its receptor LGR5, rescues the level of cell surface PAR2 from degradation by RNF43.

About the author

Rachel Bar-Shavit studied in Bar-Ilan University, Ramt-Gan, Israel and in the Weizmann Institute, Rehovot, Israel. She completed her PhD studies in Biochemistry in 1982 and her Post-Doc fellowship in Washington University Jewish Hospital St. Louis, Mo, USA in 1986. At that time  Rachel joined Sharett- Institute of oncology  at Hadassah-Hebrew University Medical Center, up till now. She is  leading a research team studying  G-Protein Coupled Receptors; (GPCRs) as  oncogenes.  Currently, acting as an Associate-Professor having a long standing interest in protease-activated receptors; PARs forming a family of 4 members (PAR1-4). The focus being on the elucidation of  stem cell  function and identification of new signaling web partners in the process of tumor invasion. A major emphasis is directed to decipher the molecular involvement of Wnt/β-catenin nuclear transcriptional activity, post translational regulation and the involvement of PAR family members in cancer. Genetically engineered cancer mouse models as also in vitro signaling assays are utilized. In-addition to outlining the molecular machinery in cancer, a major effort  is dedicated  to the development of PAR based therapy in epithelial derived tumors. Rachel and team  identified novel targets of pleckstrin-homology (PH)-binding motifs within PAR1,2&4 C-tails as a powerful platform for drug design (Kancharla A et al., Nature Communications, 2015; Nag JK & Malka H et al., Molecular Cancer Therapeutics, 2022). A lead PAR directed PH- binding motif  compound has been selected out of a library which  potently inhibits not only PAR expressing tumors but also tumors of high EGFR/erbB. It broadens the horizon of PAR based therapy also for patients that develop resistance to current therapeutic regimen.

We have identified RNF43, an E3 ubiquitin ligase as a negative regulator of PAR2 simlar to RNF43 action on  frizzleds receptors of the Wnt/β-catenin pathway (Nag JK et al., FASEB J 2023).

References

  1. Hao HX, Xie Y, Zhang Y, et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 2012;485(7397):195-200. Epub 2012/05/12.
  2. Koo BK, Spit M, Jordens I, et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature. 2012;488(7413):665-9. Epub 2012/08/17.
  3. Zebisch M, Xu Y, Krastev C, et al. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat Commun. 2013;4:2787. Epub 2013/11/15
  4. Carmon KS, Gong X, Lin Q, et al. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci U S A. 2011;108(28):11452-7. Epub 2011/06/23.
  5. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449(7165):1003-7. Epub 2007/10/16.
  6. Nag JK, Appasamy P, Sedley S, Malka H, Rudina T, Bar‐Shavit R. RNF43 induces the turnover of proteaseactivated receptor 2 in colon cancer. The FASEB Journal. 2023 Jan;37(1):e22675.

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