Citation

  • Authors: Trouvilliez S. et al.
  • Year: 2022
  • Journal: J Exp Clin Cancer Res 41 110
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: KD
    2. Name: MDA-MB-231
      Description: Human breast adenocarcinoma cells
      Known as: MDAMB231

Method

MDA-MB-231 native or KD cells were seeded (5000 cells/well; ovn, 37 °C) in a chambered coverglass system (Lab-Tek®) coated with a type I collagen matrix (100 μg/mL; Corning). These cells were transfected using the pBabe-puro-RhoA plasmid (0.15 μg; Addgene plasmid #12602) diluted in a buffer solution (JetOPTIMUS® buffer) and a transfection agent (TA; 10 min). The mixture was incorporated into the culture medium (4 h; 37 °C).

Abstract

Background: CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. Methods: After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. Results: We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. Conclusion: Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

Pubmed