Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer
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Curated by eLife
eLife assessment
The main finding in this paper is that EGFR can be a novel substrate of the membrane ZNRF3/RNF43 E3 ligases. This is significant as the prevailing understanding posits that the Wnt receptors Frizzled and LRP5/6 exclusively served as substrates for these ligases. Given the frequent occurrence of mutations in ZNRF3/RNF43 or compromised expression levels in human cancers, the new evidence that aberrant EGFR expression and signaling may also contribute to the tumorigenic effects of ZNRF3/RNF43 mutations in cancer is important. The conclusions of the manuscript are supported by solid data, but some aspects of the mechanism presented need to be reinforced to fully support the claims made by the authors.
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Abstract
ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo , whereas knockout of ZNRF3 / RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.
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eLife assessment
The main finding in this paper is that EGFR can be a novel substrate of the membrane ZNRF3/RNF43 E3 ligases. This is significant as the prevailing understanding posits that the Wnt receptors Frizzled and LRP5/6 exclusively served as substrates for these ligases. Given the frequent occurrence of mutations in ZNRF3/RNF43 or compromised expression levels in human cancers, the new evidence that aberrant EGFR expression and signaling may also contribute to the tumorigenic effects of ZNRF3/RNF43 mutations in cancer is important. The conclusions of the manuscript are supported by solid data, but some aspects of the mechanism presented need to be reinforced to fully support the claims made by the authors.
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors provide strong evidence that the cell surface E3 ubiquitin ligases RNF43 and ZNRF3, which are well known for their role in regulating cell surface levels of WNT receptors encoded by FZD genes, also target EGFR for degradation. This is a newly identified function for these ubiquitin ligases beyond their role in regulating WNT signaling. Loss of RNF43/ZNRF3 expression leads to elevated EGFR levels and signaling, suggesting a potential new axis to drive tumorigenesis, whereas overexpression of RNF43 or ZNRF3 decreases EGFR levels and signaling. Furthermore, RNF43 and ZNRF3 directly interact with EGFR through their extracellular domains.
Strengths:
The data showing that RNF43 and ZNRF3 interact with EGFR and regulate its levels and activity are thorough and convincing, …
Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors provide strong evidence that the cell surface E3 ubiquitin ligases RNF43 and ZNRF3, which are well known for their role in regulating cell surface levels of WNT receptors encoded by FZD genes, also target EGFR for degradation. This is a newly identified function for these ubiquitin ligases beyond their role in regulating WNT signaling. Loss of RNF43/ZNRF3 expression leads to elevated EGFR levels and signaling, suggesting a potential new axis to drive tumorigenesis, whereas overexpression of RNF43 or ZNRF3 decreases EGFR levels and signaling. Furthermore, RNF43 and ZNRF3 directly interact with EGFR through their extracellular domains.
Strengths:
The data showing that RNF43 and ZNRF3 interact with EGFR and regulate its levels and activity are thorough and convincing, and the conclusions are largely supported.
Weaknesses:
While the data support that EGFR is a target for RNF43/ZNRF3, some of the authors' interpretations of the data on EGFR's role relative to WNT's roles downstream of RNF43/ZNRF3 are overstated. The authors, perhaps not intentionally, promote the effect of RNF43/ZNRF3 on EGFR while minimizing their role in WNT signaling. This is the case in most of the biological assays (cell and organoid growth and mouse tumor models). For example, the conclusion of "no substantial activation of Wnt signaling" (page 14) in the prostate cancer model is currently not supported by the data and requires further examination. In fact, examination of the data presented here indicates effects on WNT/b-catenin signaling, consistent with previous studies.
Cancers in which RNF43 or ZNRF3 are deleted are often considered to be "WNT addicted", and inhibition of WNT signaling generally potently inhibits tumor growth. In particular, treatment of WNT-addicted tumors with Porcupine inhibitors leads to tumor regression. The authors should test to what extent PORCN inhibition affects tumor (and APC-min intestinal organoid) growth. If the biological effects of RNF43/ZNRF3 loss are mediated primarily or predominantly through EGFR, then PORCN inhibition should not affect tumor or organoid growth. -
Reviewer #2 (Public Review):
Using proteogenomic analysis of human cancer datasets, Yu et al, found that EGFR protein levels negatively correlate with ZNFR3/RNF43 expression across multiple cancers. Interestingly, they found that CRC harbouring the frequent RNF43 G659Vfs*41 mutation exhibits higher levels of EGFR when compared to RNF43 wild-type tumors. This is highly interesting since this mutation is generally not thought to influence Frizzled levels and Wnt-bcatenin pathway activity. Using CRISPR knockouts and overexpression experiments, the authors show that EGFR levels are modulated by ZNRF3/RNF43. Supporting these findings, modulation of ZNRF3/RNF43 activity using Rspondin also leads to increased EGFR levels. Mechanistically, the authors, show that ZNRF3/RNF43 ubiquitinate EGFR and leads to degradation. Finally, the authors …
Reviewer #2 (Public Review):
Using proteogenomic analysis of human cancer datasets, Yu et al, found that EGFR protein levels negatively correlate with ZNFR3/RNF43 expression across multiple cancers. Interestingly, they found that CRC harbouring the frequent RNF43 G659Vfs*41 mutation exhibits higher levels of EGFR when compared to RNF43 wild-type tumors. This is highly interesting since this mutation is generally not thought to influence Frizzled levels and Wnt-bcatenin pathway activity. Using CRISPR knockouts and overexpression experiments, the authors show that EGFR levels are modulated by ZNRF3/RNF43. Supporting these findings, modulation of ZNRF3/RNF43 activity using Rspondin also leads to increased EGFR levels. Mechanistically, the authors, show that ZNRF3/RNF43 ubiquitinate EGFR and leads to degradation. Finally, the authors present functional evidence that loss of ZNRF3/RNF43 unleashes EGFR-mediated cell growth in 2D culture and organoids and promotes tumor growth in vivo.
Overall, the conclusions of the manuscript are well supported by the data presented, but some aspects of the mechanism presented need to be reinforced to fully support the claims made by the authors. Additionally, the title of the paper suggests that ZNRF3 and RNF43 loss leads to the hyperactivity of EGFR and that its signalling activity contributes to cancer initiation/progression. I don't think the authors convincingly showed this in their study.
Major points:
(1) EGFR ubiquitination. All of the experiments supporting that ZNFR3/RNF43 mediates EGFR ubiquitination are performed under overexpression conditions. A major caveat is also that none of the ubiquitination experiments are performed under denaturing conditions. Therefore, it is impossible to claim that the ubiquitin immunoreactivity observed on the western blots presented in Figure 4 corresponds to ubiquitinated-EGFR species.
Another issue is that in Figure 4A, the experiments suggest that the RNF43-dependent ubiquitination of EGFR is promoted by EGF. However, there is no control showing the ubiquitination of EGFR in the absence of EGF but under RNF43 overexpression. According to the other experiments presented in Figures 4B, 4C, and 4F, there seems to be a constitutive ubiquitination of EGFR upon overexpression. How do the authors reconcile the role of ZNRF3/RNF43 vs c-cbl ?
(2) EGFR degradation vs internalization. In Figure 3C, the authors show experiments that demonstrate that RNF43 KO increases steady-state levels of EGFR and prevents its EGF-dependent proteolysis. Using flow cytometry they then present evidence that the reduction in cell surface levels of EGFR mediated by EGF is inhibited in the absence of RNF43. The authors conclude that this is due to inhibition of EGF-induced internalization of surface EGF. However, the experiments are not designed to study internalization and rather merely examine steady-state levels of surface EGFR pre and post-treatment. These changes are an integration of many things (retrograde and anterograde transport mechanisms presumable modulated by EGF). What process(es) is/are specifically affected by ZNFR3/RNF43 ? Are these processes differently regulated by c-cbl ? If the authors are specifically interested in internalization/recycling, the use of cell surface biotinylation experiments and time courses are needed to examine the effect of EGF in the presence or absence of the E3 ligases.
(3) RNF43 G659fs*41. The authors make a point in Figure 1D that this mutant leads to elevated EGFR in cancers but do not present evidence that this mutant is ineffective in mediated ubiquitination and degradation of EGFR. As this mutant maintains its ability to promote Frizzled ubiquitination and degradation, it would be important to show side by side that it does not affect EGFR. This would perhaps imply differential mechanisms for these two substrates.
(4) "Unleashing EGFR activity". The title of the paper implies that ZNRF3/RNF43 loss leads to increased EGFR expression and hence increased activity that underlies cancer. However, I could find only one direct evidence showing that increased proliferation of the HT29 cell line mutant for RNF43 could be inhibited by the EGFR inhibitor Erlotinib. All the other evidence presented that I could find is correlative or indirect (e.g. RPPA showing increased phosphorylation of pathway members upon RNF43 KO, increased proliferation of a cell line upon ZNRF3/ RNF43 KO, decreased proliferation of a cell line upon ZNRF3/RNF43 OE in vitro or in xeno...). Importantly, the authors claim that cancer initiation/ progression in ZNRF3/RNF43 mutants may in some contexts be independent of their regulation of Wnt-bcatenin signaling and relying on EGFR activity upregulation. However, this has not been tested directly. Could the authors leverage their znrf3/RNF43 prostate cancer model to test whether EGFR inhibition could lead to reduced cancer burden whereas a Frizzled or Wnt inhibitor does not?
More broadly, if EGFR signaling were to be unleashed in cancer, then one prediction would be that these cells would be more sensitive to EGFR pathway inhibition. Could the authors provide evidence that this is the case? Perhaps using isogenic cell lines or a panel of patient-derived organoids (with known genotypes).
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