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

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posted on 2025-04-03, 21:43 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

Tumor chemotherapy resistance arises frequently and limits high-grade serous ovarian cancer (HGSOC) patient survival. Focal adhesion kinase (FAK) is an intracellular protein–tyrosine kinase encoded by PTK2, a gene that is often gained in HGSOC. Canonically, FAK functions at the cell periphery. However, FAK also transits to the nucleus to modulate gene expression. We find that FAK is tyrosine-phosphorylated and nuclear-localized in tumors of patients with HGSOC surviving neoadjuvant platinum–paclitaxel chemotherapy and that FAK nuclear accumulation occurs upon subcytotoxic cisplatin exposure to ovarian tumor cells in vitro. FAK nuclear localization sequence (NLS) mutational inactivation resulted in tumor cell sensitization to cisplatin in vitro and in vivo relative to wild-type FAK-reconstituted ovarian tumor cells. Cisplatin cytotoxicity was associated with elevated ERK MAPK activation in FAK NLS− cells, cisplatin-stimulated ERK activation was also enhanced upon loss of FAK activity or expression, and cisplatin-stimulated cell death was prevented by an inhibitor of ERK signaling. MAPK phosphastase-1 (MKP1) negatively regulates ERK signaling, and cisplatin-induced MKP1 levels were significantly elevated in wild-type FAK compared with FAK NLS− ovarian tumor cells. Notably, small-molecule MKP1 inhibition enhanced both cisplatin-stimulated ERK phosphorylation and ovarian tumor cell death. Together, our results show that FAK expression, activity, and nuclear localization limit cisplatin cytotoxicity in part by regulating MKP1 levels and preventing noncanonical ERK/MAPK activation. 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.

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Keywords

Gynecological Cancers Ovarian cancer Cellular Stress Responses Cell Signaling Protein tyrosine kinases Drug Resistance Novel mechanisms

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