FAK nuclear localization enhances murine ovarian tumor cell survival to cisplatin. A, FAK-KO, FAK-WT, and FAK-NLS− KMF 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). B, Determination of cisplatin IC50 values as performed in A (*, P < 0.05; **, P < 0.01). C, FAK-WT and FAK-NLS− KMF cells were evaluated for paclitaxel cytotoxicity (μmol/L, log10) after 48 hours in culture. D, Analysis of KMF-FAK-WT and KMF-FAK-NLS− cells for growth in culture and (E) in the presence of 20 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 < 0.01). F, Representative images of crystal violet–stained KMF FAK-WT and FAK-NLS− cell colonies formed in the presence of 0, 1, or 10 μmol/L cisplatin after 10 days. G, 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 < 0.001). H, Representative images of TUNEL staining to detect changes in FAK-WT or FAK-NLS− cell DNA fragmentation after cisplatin (20 μmol/L, 48 hours) addition. TUNEL-positive nuclei (identified by DAPI costain) are marked (white circle) in the merged images. Scale bar, 10 μm. I, Quantification of FAK-WT (green bars) and FAK-NLS− (blue bars) TUNEL staining. Box and whisker plots show the mean ± SD from three independent experiments (****, P < 0.0001). ns, not significant.
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.
