Figure 6 from Nuclear Focal Adhesion Kinase Protects against Cisplatin Stress in Ovarian Carcinoma

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posted on 2024-12-20, 10:00 authored by Yichi Zhang, Marjaana Ojalill, Antonia Boyer, Xiao Lei Chen, Elise Tahon, Gaëtan Thivolle Lioux, Marvin Xia, Maryam Abbas, Halime Meryem Soylu, Douglas B. Flieder, Denise C. Connolly, Alfredo A. Molinolo, Michael T. McHale, Dwayne G. Stupack, David D. Schlaepfer

Prevention of FAK expression, activity, or nuclear localization results in elevated cisplatin-stimulated ERK activation. A, KMF FAK-WT and FAK-NLS⁻ cells were treated for 0 or 12 hours with cisplatin (20 μmol/L), and cell lysates were immunoblotted for active ERK (pERK), total ERK, and β-tubulin. B, Image quantitation of pERK to total ERK ratio from two independent experiments from A. Values are means ± SD with control FAK-WT values set to 1 (***, P < 0.001). C, Image quantitation of pERK to total ERK ratio from parental OVCAR3 and FAK-KO AB21 cells treated with cisplatin (1 μmol/L) for 24 hours. Values are means ± SD from two independent experiments with control OVCAR3 values set to 1 (*, P < 0.05). D, Representative pERK, total ERK, and GAPDH loading control immunoblotting of OVCAR3 FAK-WT and FAK-NLS⁻ lysates ± cisplatin (0.5 μmol/L, 12 hours). E, KMF parental cells were preincubated (48 hours) with FAKi (IN10018, 1 μmol/L), FAK-specific PROTAC (FAK PROTAC-1, 1 μmol/L), and FAK-Pyk2 targeting PROTAC (FAK PROTAC FC-11, 1 μmol/L) followed by with cisplatin addition (20 μmol/L, 24 hours), as indicated. Cell lysates were immunoblotted for pY397 FAK, FAK, and Pyk2 and for active (pERK) and total ERK. F, Image quantitation of pERK to total ERK ratio from three independent experiments described in E. Values are means ± SD with control set to 1 (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). G, Representative immunoblotting of OC49 ovarian PDX cell lysates for pY397 FAK, total FAK, pERK, and total ERK ± cisplatin (3.5 μmol/L, 24 hours). H, Image quantitation of pERK to total ERK ratio from two independent experiments described in G. Values are means ± SD with control set to 1 (*, P < 0.05). I, KMF FAK-WT and FAK-NLS⁻ cells were treated 20 μmol/L cisplatin (24 hours) with (DMSO) control or MEK1 inhibitor (U0125, 10 μmol/L) addition. Cell lysates were immunoblotted for active and total ERK. J, KMF FAK-WT and FAK-NLS⁻ cells were treated 20 μmol/L cisplatin (48 hours) as above with MEK1 inhibitor and cell lysates blotted for caspase-3, cleaved caspase-3, and β-tubulin. MEKi, MEK inhibitor.

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ARTICLE ABSTRACT

FAK inhibitors are in combinatorial clinical testing with agents that prevent Ras–Raf–MAPK pathway activation in various cancers. This study suggests that nuclear FAK limits ERK/MAPK activation in supporting HGSOC cell survival to cisplatin stress. Overall, it is likely that targets of FAK-mediated survival signaling may be tumor type– and context-dependent.