RNF43 inhibits WNT5A driven signaling and suppresses melanoma invasion

RNF43 is a E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also 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. This activity of RNF43 is physiologically relevant and blocks 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 inhibitor. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed 23 that RNF43 can interact with the core receptor complex components dedicated to the 24 noncanonical Wnt pathway such as ROR1, ROR2, VANGL1 and VANGL2. RNF43 triggers 25 VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of 26 ROR1 receptor. This activity of RNF43 is physiologically relevant and blocks pro-metastatic 27 WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the 28 suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-29 assisted development of resistance to BRAF V600E inhibitor. In line with these findings, RNF43 30 expression decreases during melanoma progression and RNF43-low patients have worse 31 prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-32 mediated biological responses that desensitizes cells to WNT5A. 33 Introduction cells deficient in both RNF43 and ZNRF3 (RNF43/ZNRF3 dKO; R/Z dKO) showed higher 163 VANGL2 levels and higher DVL phosphorylation (Fig. 3B, Figure 3 figure supplement 1B). 164 Interestingly, treatment with proteasome inhibitor MG132 but not with autophagosome-lysosome 165 inhibitor Chloroquine blocked these effects of RNF43 (Fig. 3C). This suggests that RNF43 action 166 in noncanonical Wnt pathway depends on the proteasomal degradation pathway, which differs 167 from the Wnt/β-catenin pathway, where RNF43 triggers FZD degradation via lysosomal pathway 168

Vemurafenib. 292
Therefore, we challenged with Vemurafenib A375 WT and its RNF43 expressing 293 derivatives. As shown in Fig. 6E exogenous RNF43 decreased colony formation and proliferation 294 of cells seeded in the low density and Vemurafenib further enhanced this effect. Importantly, both 295 A375 WT and A375 IV overexpressing RNF43 completely failed to develop resistance to 296 Vemurafenib and died off during the selection at 1 μM Vemurafenib concentration (Fig. 6F). 297 Altogether these data confirm earlier findings on the importance of WNT5A signaling in the 298 acquisition of Vemurafenib resistance and demonstrate that RNF43 can completely block this 299 process. 300

Discussion 302
Our study identified RNF43 as the inhibitor of noncanonical WNT5A-induced signaling. 303 RNF43 physically interacted with multiple receptor components of the Wnt/PCP pathway such as 304 ROR1/2, VANGL1/2 or DVL1/2/3 and triggered degradation of VANGL2 and membrane clearance 305 of ROR1; ultimately resulting in the reduced cell sensitivity to WNT5A. The newly discovered 306 RNF43 action in WNT5A-mediated signaling seems to be mechanistically different than the well-307 known function in the Wnt/β-catenin pathway. For example, we observed ROR1 and VANGL2 308 interaction with RNF43 in the absence of DVL. In contrast, DVL seems to be essential for the 309 inhibition further highlights importance of WNT5A signaling in this process and also uncovers a 343 mechanism that can be explored therapeutically. 344 Relevance of our findings is likely not limited to melanoma. Signaling cascade RSPO-345 LGR4/5-RNRF43/ZNRF3 has been shown to regulate variety of biological processes. In light of 346 our results, it is tempting to speculate that WNT5A-RNF43 axis regulates other developmental, 347 physiological and patho-physiological conditions. For example, WNT5A is overexpressed in   by the global minimum and e) differential expression using LIMMA statistical test. Prior to volcano 503 plot plotting, suspected BirA* binders were filtered out (proteins identified on at least 2 peptides 504 in both technical replicates of particular BirA* sample, and present in >3 samples). Volcano plot 505 was created in R using ggplot2 and ggrepel R packages by R version 3.6.1. Proteins with adjusted 506 p-value <0.05 and log fold change >1 were further subjected to gene ontology tools, considering 507 only the first ID of majority protein IDs: g:Profiler online tool (https://biit.cs.ut.ee/gprofiler/gost,

Western blotting and antibodies 535
Western blot analysis was performed as it was described before using samples with same 536    Table 3. 608

Colony formation assay 642
To assess an ability to colony formation in the presence of 0.3 μM vemurafenib, 300 of After that time, medium was removed and colonies were washed in PBS, fixed in the ice-cold 645 methanol for 30 min and stained in the 0.5% crystal violet in 25% methanol. After washing and 646 drying, bound crystal violated was eluted with 10% acetic acid and absorbance at 590 nm was 647 measured on Tecan Sunrise plate reader. Result were normalized to the non-treated A375 wild 648 type results. 649 650