Citation

  • Authors: Winter M. et al.
  • Year: 2021
  • Journal: Cells 10 1504
  • Applications: in vitro / DNA, siRNA / INTERFERin, jetOPTIMUS
  • Cell type: MDA-MB-231
    Description: Human breast adenocarcinoma cells
    Known as: MDAMB231

Method

Native MDA-MB-231 cells (100,000) were seeded in 6-well plates. After 24 h, cells were transfected with 1 µg/mL of plasmids purchased from Addgene: the pVimentin-PSmOrange-N1 (#31922) contains vimentin; the pPSmOrange-N1 (#31898) was used as control. Transfection was performed by using jetOPTIMUS transfection reagent following the manufacturer’s instructions (Polyplus transfection). Experiments were performed 24 h after transfection, and vimentin overexpression was validated by using fluorescent microscopy with pPSmOrange fluorescence (Nikon, 10×) as previously described. MDA-MB-231 persistent cells (50,000) were seeded in 6-well plates and then transfected with 100 nM siRNA using INTERFERin™ transfection reagent following the manufacturer’s instructions (Polyplus Transfection®). Two sequences of siRNA against vimentin (siVIM5, S100302197; siVIM13, S104201890) as well as control (siCTL, S103650325) were from Qiagen. Experiments were performed 72 h after siRNA transfection.

Abstract

Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.

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