Figure 6. DDR2 phosphorylation sensitizes YAP/TAZ mediated ferroptosis through Src activity.

(A) DDR2 upregulation in recurrent tumor cells was further enhanced by erastin. Western blots measured DDR2 levels in two primary and two recurrent tumor cell lines before or after erastin treatment (0.5 μM, 16 hours). (B) DDR2 knockdown abolished basal and enhanced DDR2 protein expression. Recurrent tumor cells transduced with Ddr2 shRNA were treated with erastin (0.5 μM, 16 hours) for Western blots. (C) RT-PCR showed the upregulation of Ddr2 mRNA by erastin treatment (0.5 μM, 16 hours) in recurrent tumor cells. (D) Erastin treatment increased DDR2 phosphorylation. The protein lysate of recurrent tumor cells treated with DMSO or erastin (0.5 μM, 16 hours) were immunoprecipitated by the pan-phospho-Ser/Thr (pSpT) antibody and blotted with DDR2 antibody for phosphorylated DDR2. (E) Erastin triggered phosphorylation of DDR2 protein at Y740. Recurrent tumor cells treated with erastin (0.5 μM) and DMSO, dasatinib (1 μM), or saracatinib (1 μM) for 16 hours were stained with phospho-DDR2(Y740) antibody for DDR2 phosphorylation and activation. (F) Collagen I coating increased ferroptosis sensitivity by DDR2. Recurrent tumor cells transduced with Ddr2 shRNA were plated on a regular or collagen I coating plate for 19 hours of erastin treatment and CellTiter Glo assay. (G) The constitutively active mutant of DDR2 (Y740F) increased the expression of YAP/TAZ targeted gene, CTGF. The CTGF RNA expression in T47D cells overexpressing empty vector, DDR2 wild type cDNA, or constitutively active mutant of DDR2 (Y740F) was determined by RT-PCR. (H) DDR2 Y740F mutant increased ferroptosis sensitivity. The erastin sensitivity of T47D cells in (G) were tested using CellTiter Glo assay. (I-J) Pharmacological suppression of DDR2, Src, and YAP decreased the CTGF upregulation and ferroptosis sensitivity promoted by DDR2 Y740F cDNA. T47D cells overexpressing DDR2 Y740F cDNA were treated with DMSO, dasatinib (1 μM, DDR2 inhibitor), saracatinib (1 μM, Src inhibitor), or verteporfin (2 μM, YAP inhibitor). CTGF expression was determined after 18 hours of incubation by RT-PCR in (I). Ferroptosis sensitivity was determined by co-treatment with erastin for 24 hours and CellTiter Glo assay in (J). (K) Pharmacological suppression of DDR2, Src, and YAP prevented recurrent tumor cells from erastin-induced ferroptosis. Recurrent tumor cells under erastin treatment were co-treated with DMSO, dasatinib (1 μM), saracatinib (1 μM), or verteporfin (2 μM) for 18 hours for CellTiter Glo assay. (L) Schematic illustration of EMT-driven DDR2 upregulation in determining ferroptosis through the regulation of YAP/TAZ. (F, H, J, K) n=3 biological replicates. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Dunnett’s multiple comparisons. Bars show S.E.M.