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. Significance: 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.

Background: Ovarian cancer is an immunologically cold tumor that is treated with surgery and a chemotherapy regimen of platinum agents with taxanes. Paradoxically, elevated levels of several immune markers are effective at predicting prognosis for patients with ovarian cancer, though it is not clear how chemotherapy might influence this. Chemotherapy elicits immunogenic cell death, yet tumor-controlling doses of chemotherapy are also immunotoxic. Objectives: To evaluate interactions of chemotherapy with the immune system, we studied the impact of chemotherapy in an aggressive mouse model of ovarian cancer developed within our lab. Methods: Using a single-cell transcriptomics sequencing approach, supported by flow cytometry, we evaluated the influence of a first-line therapy, cisplatin and docetaxel, and a second-line therapy, pegylated liposomal doxorubicin (PLD), on control of tumor growth and on tumor-associated immune populations of cells. Results: Both chemotherapy approaches were effective at controlling tumor growth and selectively depleted tumor cells from distinct transcriptional clusters. Both chemotherapies also resulted in relative increases in immune populations compared to untreated tumor-bearing mice, but immune populations from PLD-treated mice were more abundant and expressed a greater fraction of maturity-associated transcripts and increased proportions of tumor resident macrophage populations. PLD treatment selectively upregulated MHC class II on tumor cells, and this could be replicated in vitro across ovarian cancer cell lines and in patient tumor cells ex vivo. Conclusions: Altogether, the results support the notion that PLD has a greater capacity for immunopotentiation, which may be important to consider if immunotherapy approaches are adapted for ovarian tumors in the future.

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.

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.

Active FAK staining increases in the nucleus of HGSOC patient tumors after neoadjuvant chemotherapy. A, IHC staining of paraffin-embedded serial initial tumor biopsy sections (patient 1014109 and patient 3000276) and corresponding tumor resection after neoadjuvant chemotherapy treatment. Samples were stained with H&E and phosphospecific antibodies to the FAK activation loop Y576 (pY576) within the kinase domain. Scale is 100 μm (inset scale is 25 μm). B, Image quantification of percent of tumor cell area exhibiting FAK pY576 staining. Paired tumor samples (n = 8) from initial biopsy (blue circles) and from surgical tumor debulking after neoadjuvant chemotherapy (red circles). Dotted lines connect paired patient tumor samples collected prior to and after neoadjuvant chemotherapy (***, P < 0.001). Chemo, chemotherapy; H&E, hematoxylin and eosin; NeoAdj, neoadjuvant.

Nuclear FAK regulation of MKP1 expression is associated with cisplatin resistance. A, KMF FAK-WT and FAK-NLS− cells were treated with increased concentrations of cisplatin (0, 10, or 20 μmol/L) for 12 hours and protein lysates were immunoblotted for GFP-FAK, MKP1, pERK, and total ERK. B, Image quantitation of pERK to total ERK ratio or MKP1 levels (FAK-WT set to 0) from two independent experiments described in A. Values are means ± SD (***, P < 0.001). C, KMF FAK-WT and FAK-NLS− cells were treated 20 μmol/L cisplatin (24 hours) under control (DMSO) or with MKP1 inhibitor (BCI-215, 1 μmol/L) addition. Protein lysates were immunoblotted for pERK and total ERK. D, Image quantitation of pERK to total ERK ratio from two independent experiments described in C. Values are means ± SD with FAK-WT control set to 1 (*, P < 0.05). E, KMF FAK-WT and FAK-NLS− cells were treated 20 μmol/L cisplatin (48 hours) as above with MKP1 inhibitor (BCI-215, 1 μmol/L) addition, and cell lysates were immunoblotted for caspase-3 and cleaved caspase-3. F, Determination of IC50 values to cisplatin KMF FAK-WT (green bars) and FAK-NLS− (blue bars) cells in the presence of MKP1 inhibitor (BCI-215, 1 μmol/L) or DMSO control as determined by alamarBlue cell viability assay. Values are means ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). G, Kaplan–Meier curves from 949 HGSOC patient samples that received adjuvant carbo- or cisplatin-based chemotherapy. Plots show probability of relapse-free survival in months with tumors high (red) or low (black) for MKP1 mRNA (HR = 1.4, P = 0.0024). ns, not significant; WCL, whole-cell protein lysate.