To observe the impact of NF2 and YAP expression on ferroptosis of triple negative breast cancer cells with high expression of CD24 and explore the related molecular mechanism.

MDA-MB-231 breast cancer cells with high expression of CD24 (CD24^high) and low expression of CD24 (CD24^low) were cultured. The cells were treated with ferroptosis agonists Erastin, RSL3 and cysteine starvation. The cell death was detected by Trypan blue staining (TBA). Malondialdehyde(MDA)content was measured using the TBA method to analyze the level of cellular ferroptosis. Western blot analysis was used to detect the protein expression of NF2 and YAP in CD24^high and CD24^low MDA-MB-231 cells. Lentiviral vectors were used to knock out and over-express NF2 and YAP, and changes in cellular ferroptosis were observed. The real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of fibroblast-specific protein 1(FSP1)after NF2 knock out and YAP over-expression.

After treatment with cysteine starvation, the ferroptosis inducers Erastin and RSL3, the death rate of MDA-MB-231 cells increased, with the death rate of CD24^low cells significantly higher than that of CD24^high cells (t=14.548, P<0.001; t=8.310, P=0.001; t=8.600, P=0.001). Meanwhile, the TBA method results showed that there was a significant difference in MDA content between CD24^high and CD24^low MDA-MB-231 cells under conditions with or without cysteine (t=-3.920, P=0.017; t=11.566, P<0.001); compared with the control group with DMSO, the MDA levels of CD24^high and CD24^low cells significantly increased after treatment with the ferroptosis inducers Erastin and RSL3, with the MDA content of CD24^low cells significantly higher than that of CD24^high cells (t=10.763, P=0.006; t=24.067, P<0.001). Western blot experiments showed that there was a significant difference in the protein content of NF2 and YAP between CD24^low and CD24^high MDA-MB-231 cells (t=-4.331, P=0.012; t=4.219, P=0.013), with higher expression of YAP protein in CD24^low cells (t=4.219, P=0.013) and higher expression of NF2 protein in CD24^high cells (t=-4.331, P=0.012). NF2 knockout or (and) YAP overexpression cell lines were constructed in CD24^high cells, and real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of the ferroptosis key protein FSP1 among the four groups (F=30.297, P<0.001). Meanwhile, the ferroptosis situation of CD24^high MDA-MB-231 cells after NF2 knockout or (and) YAP overexpression was detected, and the results showed that knocking out NF2 or (and) overexpressing YAP could promote cell death to a certain extent after adding the ferroptosis inducer Erastin (F=38.911, P<0.050), with the highest death rate of CD24^high MDA-MB-231 cells under the condition of NF2 knockout combined with YAP overexpression. The addition of the ferroptosis inhibitor Fer-1 after Erastin led to a decrease in the death rate of cells, with no statistically significant difference among the four groups (F=0.256, P=0.855).

Knocking out NF2 and overexpressing YAP in CD24^high triple negative breast cancer cells can inhibit the expression of FSP1 and promote cell ferroptosis.