RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy
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Evaluation Summary:
Radaszkiewicz and collaborators describe RNF43 as a novel negative regulator of WNT5A-induced signaling in human cells. They demonstrate that RNF43 can interact with proteins in this pathway, namely ROR1, ROR2, VANGL1 and VANGL2. Specifically, they find that, through these interactions, RNF43 can suppress invasive properties of melanoma cells, as well as the development of resistance to BRAF V600E inhibitor. The experiments are well done and well explained; however, they were performed only in an in vitro setting and with a very limited number of cell lines.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.
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Reviewer #3 (Public Review):
The authors herein have nicely dissected the role of RNF43 in WNT5A signaling in mammalian cells, with a focus in the context of melanoma. They show that RNF43 inhibits WNT5A activity by ubiquitinating and thereby marking for proteasomal degradation multiple proteins involved in WNT5A signal transduction (i.e., VANGL2). The authors have performed the study in a thorough manner.
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Reviewer #2 (Public Review):
In the present manuscript Radaszkiewicz et al. analyze the role of Ring Finger Protein 43 (RNF43) in inhibiting the noncanonical WNT5A pathway. The authors demonstrate that RNF43 can interact with proteins involved in the WNT5A pathway, including ROR1, ROR2, VANGL1 and VANGL2. Specifically, they propose that RNF43 induces: i) VANGL2 ubiquitination and proteasomal degradation and ii) clathrin-dependent internalization of the ROR1 receptor. Considering the role of the WNT5A pathway in melanoma metastasis and resistance to targeted therapy, the authors further explore the role of RNF43 in melanoma invasion and resistance to vemurafenib. The authors ultimately conclude that RNF43 can prevent invasion and resistance to targeted therapy by inhibiting the WNT5A pathway. The data supporting the interaction between …
Reviewer #2 (Public Review):
In the present manuscript Radaszkiewicz et al. analyze the role of Ring Finger Protein 43 (RNF43) in inhibiting the noncanonical WNT5A pathway. The authors demonstrate that RNF43 can interact with proteins involved in the WNT5A pathway, including ROR1, ROR2, VANGL1 and VANGL2. Specifically, they propose that RNF43 induces: i) VANGL2 ubiquitination and proteasomal degradation and ii) clathrin-dependent internalization of the ROR1 receptor. Considering the role of the WNT5A pathway in melanoma metastasis and resistance to targeted therapy, the authors further explore the role of RNF43 in melanoma invasion and resistance to vemurafenib. The authors ultimately conclude that RNF43 can prevent invasion and resistance to targeted therapy by inhibiting the WNT5A pathway. The data supporting the interaction between RNF43 and proteins involved in the WNT5A pathway are pretty rigorous. However, the study would benefit from additional experiments in the context of RNF43's role in invasion and resistance to targeted therapy in melanoma. Overall, the techniques utilized in the manuscript are appropriate, however additional cell lines and in vivo studies are strongly recommended to strengthen the manuscript.
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Reviewer #1 (Public Review):
The authors present data suggesting that RNF43 affects WNT5a signaling through turnover of ROR1 and ROR2 receptors on the cell surface. The strengths of this work are the many overexpression, knockdown and mutant cell lines the authors use to delineate specific protein interactions and localizations. The authors have done a good job of analyzing the interaction of multiple proteins within the Wnt signaling pathways to determine how RNF43 affects expression of proteins associated with non-canonical Wnt signaling. The weakness of this study is that most of these protein interactions were performed in 293 cells and not in melanoma cell lines. One melanoma cell line was used to relate the protein interactions studied in 293 cells to signaling in melanoma. The authors present data that suggest RNF43 decreases …
Reviewer #1 (Public Review):
The authors present data suggesting that RNF43 affects WNT5a signaling through turnover of ROR1 and ROR2 receptors on the cell surface. The strengths of this work are the many overexpression, knockdown and mutant cell lines the authors use to delineate specific protein interactions and localizations. The authors have done a good job of analyzing the interaction of multiple proteins within the Wnt signaling pathways to determine how RNF43 affects expression of proteins associated with non-canonical Wnt signaling. The weakness of this study is that most of these protein interactions were performed in 293 cells and not in melanoma cell lines. One melanoma cell line was used to relate the protein interactions studied in 293 cells to signaling in melanoma. The authors present data that suggest RNF43 decreases invasion and proliferation of melanoma cells in vitro. Analyzing the role of RNF43 in invasion, proliferation and signaling in more than one melanoma cell line would strengthen the authors conclusions about the role of RNF43 in Wnt5A signaling in melanoma.
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Evaluation Summary:
Radaszkiewicz and collaborators describe RNF43 as a novel negative regulator of WNT5A-induced signaling in human cells. They demonstrate that RNF43 can interact with proteins in this pathway, namely ROR1, ROR2, VANGL1 and VANGL2. Specifically, they find that, through these interactions, RNF43 can suppress invasive properties of melanoma cells, as well as the development of resistance to BRAF V600E inhibitor. The experiments are well done and well explained; however, they were performed only in an in vitro setting and with a very limited number of cell lines.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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