Fig. 10: Uncoupling PEAK1 from AXL signaling leads to impaired metastasis.

a Lysates of rescued MDA-MB-231-Luc CRISPR PEAK1 knockout cells. b Representative in vivo bioluminescent images of fat pad-injected mice with MDA-MB-231-Luc PEAK1 knockouts rescued with EV, PEAK1 WT, or PEAK1 3PA mutant 8 and 7 weeks postinjection, respectively. c Bioluminescence quantification of tumor growth at different weeks postinjection of mice bearing PEAK1 KO rescued with EV (n = 8), PEAK1 WT (n = 8), or PEAK1 3PA (n = 8). d Bioluminescence quantification of the spontaneous metastases in the lungs measured ex vivo of the fat pad-injected mice shown in c when the tumors from PEAK1^WT and PEAK1^3PA reached the same size (7–8 weeks postinjection). *P = 0.0299; **P = 0.0125. n = 8 for EV, n = 8 for PEAK1 WT, and n = 8 for PEAK1 3PA. e Circulating tumor cells isolated from mice-bearing EV PEAK1^KO, PEAK1^WT, or PEAK1^3PA rescued mammary tumors. *P = 0.0098, ***P = 0.0003 (n = 8 for each group). f Representative confocal images of parental MDA-MB-231 cells and PEAK1 KO cells rescued with EV, PEAK1 WT, or PEAK1 3PA. Cells were stained for PXN (green) and Phalloidin (magenta). Scale bar, 10 μm. g Quantification of the FA number per cell depicted in f. *P = 0.014; ****P < 0.000001 (n = 3 experiments with 30 cells per condition). h Quantification of the size of the adhesions quantified in g. ****P < 0.000001 (n = 3 experiments with 30 cells per condition). Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value, and the lower whisker extends from the hinge to the smallest value (g, h). Two-tailed Mann–Whitney test was used to calculate P value. Source data are provided as a Source data file. i Schematic model of AXL and EGFR signaling in a cancer cell, where EGFR modulates adherens junctions and AXL modulates the FA turnover. Upon EGF stimulation, EGFR induces the expression of MMPs to degrade the adherens junctions. Once cell–cell contact is lost, AXL activation by GAS6 or EGFR can lead to the modulation of FAs turnover at the rear or front end of the cell. In specific, AXL directly phosphorylates NEDD9 to recruit its complex formation with PTK2 (FAK) and CRKII. Simultaneously, AXL can phosphorylate PEAK1, in complex with PRAG1 and CRKII, and recruit PTK2/NEDD9/CRKII/PEAK1/PRAG1 to PXN-positive FAs. Upon PEAK1 phosphorylation, CSK is recruited to PEAK1/PRAG1 complex at FAs to induce tyrosine phosphorylation of PXN. In addition, PEAK1 can also recruit βPIX/GIT1 complex to PXN-positive FAs to induce FA disassembly and turnover.