Figure 4 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

FAK KO and reexpression show that nuclear FAK promotes cisplatin but not paclitaxel resistance in human OVCAR3 cells. A, Immunoblots of cultured FAK KO OVCAR3 (clone AB21) and GFP-FAK-WT or FAK-NLS− reconstituted AB21 cells for HA-tag (at FAK C-terminal), FAK pY397, and β-tubulin as a loading control. B, FAK-WT, and FAK-NLS− OVCAR3 cells were evaluated for cisplatin cytotoxicity after 48 hours in culture. Shown is percent cell viability vs. cisplatin concentration (μmol/L, log10), and points are means of triplicate samples ± SD (n = 3 independent experiments). C, Determination of IC50 values to cisplatin as performed in B (***, P < 0.001). D, FAK-WT and FAK-NLS− OVCAR3 cells were evaluated for paclitaxel cytotoxicity (μmol/L, log10) after 48 hours in culture. E, Analysis of OVCAR3 FAK-WT and FAK-NLS− cells for growth in culture and (F) in the presence of 0.5 μmol/L cisplatin for 72 hours. Values are means ± SD from two independent experiments with triplicate points and FAK-WT values set to 1 (***, P < 0.001; ns not significant). G, Representative images of crystal violet–stained OVCAR3 FAK-WT and FAK-NLS− cell colonies formed in the presence of 0, 0.5, or 1.0 μmol/L cisplatin after 10 days. H, Quantitation of FAK-WT (green bars) and FAK-NLS− (blue bars) colony formation. Values are means ± SD from three independent experiments with triplicate points (*, P < 0.05).

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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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