New Tool Maps How Cancer Cells Hijack Growth Signals
Researchers developed a faster way to measure how Wnt proteins bind to cell receptors in colorectal cancer, potentially accelerating drug discovery. The technique could help pharmaceutical companies identify promising cancer therapies more efficiently by revealing exactly how these critical molecular interactions work.
Originaltitel: NanoBiT- and NanoBiT/BRET-based assays allow the analysis of binding kinetics of Wnt-3a to endogenous Frizzled 7 in a colorectal cancer model
<p> Background and Purpose</p><p>Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD<sub>7</sub>, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer.</p><p>Experimental Approach</p><p>SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD<sub>7</sub>, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD<sub>7</sub> using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization.</p><p>Key Results</p><p>With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD<sub>7</sub> was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated <em>K</em><sub>d</sub>. Thus, measurements of binding affinities to FZD<sub>7</sub> obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells.</p><p>Conclusions and Implications</p><p>Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD<sub>7</sub> binding should be performed using receptors expressed under endogenous promotion.</p>