American Association for Cancer Research
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Supplementary Figure 2 from Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1–Dependent Trafficking of Integrins

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posted on 2023-04-03, 17:44 authored by Polina Y. Kozyulina, Yuriy V. Loskutov, Varvara K. Kozyreva, Anuradha Rajulapati, Ryan J. Ice, Brandon C. Jones, Elena N. Pugacheva

Supplementary Figure 2. (A) Quantification of relative fluorescence intensity units (RFU) of ß1 integrin in BT459 and MDA-MB231LN cells as in (B); graph is mean RFU, % of siCon (assigned as 100%) +/-S.E.M; n=3, 15 cells/per treatment; one-way ANOVA with Dunnett's post-hoc analysis; ligand-bound: MDA-MD-231LN *p<0.023 (siCon/siN2 and N3), BT459 *p<0.0052 (siCon/siN2 and N3); ligand-free: p is not significant (ns). (B). Representative confocal images of MDA-MB-231 cells treated with siCon and siNEDD9 (N3) for 48h and stained with ß1 integrin (ligand-bound/extended and ligand-free/bent) (green) without permeabilization, DNA (blue); Dotted lines represent cell borders, insets-the enlarged areas of individual cells (white lines) of b1 integrin surface staining (left) or heatmaps of b1 integrin surface staining intesity (right) indicated by rectangles in the main panel. Scale bar, 10$m; (C). Quantification of relative fluorescence intensity units (RFU) of ligand-bound ß1 integrin in MDA-MB-453 and ZR75-1 cells with or w/o Nedd9-cDNA; graph is mean RFU, % of control (assigned as 100%) +/-S.E.M; n=3, 15 cells/per treatment; t-test: MDA-MB-453 and ZR75-1 *p<0.0001 (w/o Nedd9-cDNA/with Nedd9-cDNA). (D). Quantification of number of FITC-Col.I positive vesicles (%) co-stained with EEA1 or Rab7 in MDA-MB-435 cells cells incubated with FITC-collagen I and stained with anti-EEA1/or Rab7; normalized to control; 15cells/per staining; t-test, EEA1: *p<0.0001(w/o Nedd9-cDNA/with Nedd9-cDNA), Rab7: *p=0.0088 (w/o Nedd9-cDNA/with Nedd9-cDNA). (E-F). Confocal images of MDA-MB-231-siCon,-siN2) cells stained with anti-Integrin b1 active (12G10)



The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand–integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity.Implications: This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins. Mol Cancer Res; 13(3); 423–38. ©2014 AACR.