Nuclear FAK accumulation occurs upon cisplatin treatment of human and murine ovarian tumor cells. A, Nuclear and cytoplasmic cell fractionation of human OVCAR4 cells treated with DMSO (control) or cisplatin (1 μmol/L) for 12 hours followed by immunoblotting for FAK pY397, total FAK, β-tubulin (cytoplasmic marker), and histone H3 (nuclear marker). B, Schematic of GFP fusion to FAK denoting N-terminal band 4.1, ezrin, radixin, moesin homology (FERM), kinase, and focal adhesion targeting (FAT) domains. FAK FERM arginine R177 and R178 to alanine mutational changes prevent FAK nuclear localization. C, Stable reexpression of GFP-FAK-WT (WT) and GFP-FAK-NLS− (NLS−) in KMF FAK KO murine ovarian tumor cells as analyzed by FAK, pY397 FAK, and β-actin immunoblotting. D, KMF FAK-WT and KMF FAK-NLS− cells treated with DMSO (control) or cisplatin (20 μmol/L) for 1 hour and immunoblotting for FAK pY397 and total FAK. E, Confocal immunofluorescence imaging was used to visualize GFP-FAK and DAPI stain in FAK-WT and FAK-NLS− cells treated with DMSO (control) or cisplatin (20 μmol/L) for 12 hours. Shown are representative images of GFP-FAK (images shown at midline of Z-stack), nucleus, and merged images. Scale bar, 10 μm. F, Nuclear and cytoplasmic fractionation of FAK-WT and FAK-NLS− cells treated with cisplatin (20 μmol/L) for 0, 12, or 24 hours followed by immunoblotting for FAK, β-tubulin, and histone H3. G, Representative images of GFP-FAK-WT and FAK-NLS− expressing (images shown at midline of Z-stack) and DAPI-stained cells treated with leptomycin B (50 nmol/L) for 6 hours. Scale bar, 10 μm. Cyto, cytoplasmic.
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.
