Article Figures & Data
Figures
- Figure 1.
Disruption of Cdc37 chaperone binding to FAK mimics FAK kinase inhibition. A, IP of FAK from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cell lysates, immunoblotted for FAK, Cdc37, and phosphorylated Cdc37 (S13). B, IP of FAK from SCC FAK-WT cells treated with DMSO (0.1%) or 250 nmol/L VS-4718, VS-6063, PF-562,271, or GSK2256098 for 24 hours, immunoblotted for FAK, phosphorylated FAK (Y397), and Cdc37. C, Lysates from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cells, immunoblotted for FAK and phosphorylated FAK (Y397). D, IP of FAK from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cell lysates, immunoblotted for FAK and Src. E, SCC FAK-WT cells seeded on glass coverslips, fixed, and labeled with anti-FAK (green), anti-Cdc37 (red), and DAPI (blue). Inset (right) represents dashed boxed region of main image. Arrows and arrowheads (inset) indicate examples of regions of colocalization of FAK and Cdc37. Scale bar, 20 μm. F, SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cells seeded on glass coverslips, fixed, and labeled with anti-FAK (green) and DAPI (blue). Scale bars, 20 μm. G, FAK synthesis and degradation profiles quantified by metabolic labeling and mass spectrometry. FAK-WT (green) and FAK-G431A,F433A (brown) synthesis and degradation curves were determined from normalized SILAC ratio profiles by nonlinear regression and plotted as means ± SEM with best fit curves and 95% confidence interval bands (n ≥ 5 peptides; representative of three independent experiments). NS, not significant (extra sum-of-squares F test). Bar chart inset summarizes inferred 50% protein turnover times for FAK-WT and FAK-G431A,F433A, plotted as means ± SD (n = 3 independent experiments). NS, not significant (two-tailed Mann–Whitney U test). H, Label-free mass spectrometric characterization of FAK-interacting proteins isolated by IP of FAK from SCC FAK-WT and FAK-G431A,F433A cell lysates. Specific FAK-binding proteins were determined versus IP from SCC FAK−/− cells (n = 3 independent experiments), satisfying Q < 0.05 (Student t test with permutation-based FDR correction). Gray curves show the threshold for significant differential regulation (FDR, 5%; artificial within-groups variance, 1). Proteins satisfying P < 0.01 and fold change > 4 are labeled with gene names for clarity. I, Label-free mass spectrometric characterization of FAK-interacting proteins isolated by IP from SCC FAK-WT, FAK-KD, and FAK-G431A,F433A cell lysates and lysates from SCC FAK-WT cells treated with 250 nmol/L VS-4718 (n = 3 independent experiments). Normalized label-free quantification of protein abundance for each protein was converted to a Z-score. Differentially regulated, specific FAK-binding proteins satisfying Q < 0.05 (one-way ANOVA with permutation-based FDR correction) were hierarchically clustered and displayed as a heatmap. Proteins are labeled with gene names for clarity.
- Figure 2.
Identification of synergistic combinations with FAK kinase inhibitors. A, Summary of compound screen to identify drug sensitivity in the presence of reduced FAK kinase activity. To identify differential phenotypic effects between FAK-WT and FAK-mutant (FAK-G431A,F433A or FAK−/−) cells, for each image-based measurement Z-score, the FAK-WT value was subtracted from each FAK mutant value. Differential phenotypic effects were analyzed by principal component analysis. Principal components (PC) 1 and 2 account for 51.3% and 22.1% of the total variance, respectively. Cellular features used in the phenotypic analysis are listed in the inset (right). B, Hit compounds identified in the phenotypic screen. Vorinostat is labeled as SAHA in A and B. C, SCC FAK-WT, FAK-G431A,F433A, and FAK−/− cells treated with 10 μmol/L vorinostat. Blue, nuclei; green, F-actin; red, HCS Cell Mask. Scale bar, 50 μm. D, Combination of vorinostat (top) or panobinostat (bottom) with VS-4718 inhibits spheroid growth. E, Dose matrices of spheroid area following treatment with vorinostat (top) or panobinostat (bottom) in combination with VS-4178 for 7 days. F, Quantification of RFP (G1 phase; top) and GFP (G2–M phase; bottom) FUCCI cell-cycle reporter expression in SCC FAK-WT spheroids treated with vorinostat (left) or panobinostat (right) for 7 days. For combination treatments with VS-4718, 500 nmol/L VS-4718 was used. RFI, relative fluorescence intensity. G, SCC FAK-WT spheroids expressing FUCCI cell-cycle reporter were treated for 7 days with vorinostat (Vorino.; 1.25 μmol/L) or panobinostat (Pano.; 6.25 nmol/L) in combination with VS-4718 (500 nmol/L). For D–F, data are normalized to DMSO values and are displayed as means ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA). H, Lysates from SCC FAK-WT spheroids treated with VS-4718, vorinostat (Vorino.), or panobinostat (Pano.) for 24 hours and immunoblotted for FAK, phosphorylated FAK (Y397), histone H3, and acetylated (K-Ac) histone H3. I, Combination of panobinostat and VS-4718 inhibits SCC FAK-WT tumor growth. Mice were treated with drug(s) from day 0. Group tumor volumes are displayed as mean ± SEM [n = 5 mice/group (2 tumors/mouse)]. ***, P < 0.001 (one-way ANOVA).
- Figure 3.
Identification of cells that are sensitive to FAK and HDAC inhibitor combinations. A, Nuclei counts of A549 (left) and Flo1 (right) cells following treatment with vorinostat (5 μmol/L), panobinostat (7.5 nmol/L), and VS-4718 (500 nmol/L) for 24 hours. Data are normalized to DMSO values and are displayed as mean ± SEM (n = 2 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA). B, Combined inhibition of FAK and HDAC blocks colony formation. Representative images are shown (top). Bar charts (bottom) are plotted as means ± SEM (n ≥ 3 independent experiments). C, Combination of FAK and HDAC inhibition reduces cell viability. Dose matrices of viability of A549 (top) and Flo1 (bottom) cells following treatment with vorinostat (μmol/L; left) or panobinostat (nmol/L; right). D, Cell-cycle distribution in A549 (left) and Flo1 (right) cells following treatment with the same drugs as used in B for 24 hours. Results in C and D are displayed as means (n ≥ 3 independent experiments). E, FAK and HDAC inhibition enhances the induction of apoptosis in A549 (left) and Flo1 (right) cells. Bar charts are plotted as means ± SEM (n ≥ 3 independent experiments). F, Combination of panobinostat and VS-4718 inhibits A549 (left) or Flo1 (right) tumor growth. Tumor volumes are plotted as means ± SEM [n ≥ 4 mice/group (2 tumors/mouse)]. Mice were treated from day 0 and accompanying body weights are shown (mean ± SD). For B, E, and F, *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA).
- Figure 4.
Combined inhibition of HDAC and FAK abolishes FAK kinase activity and regulates YAP localization. A, Lysates from A549 (left) and Flo1 (right) cells treated as indicated were immunoblotted for FAK, phosphorylated FAK (Y397), GAPDH, histone H3, and acetylated (K-Ac) histone H3. Quantification of phosphorylated FAK (Y397) immunoblotting (bottom) is displayed as means ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01 (one-way ANOVA). B, Lysates from A549 (left) and Flo1 (right) cells were immunoblotted for YAP, phosphorylated YAP (S127), FAK, phosphorylated FAK (Y397), and GAPDH at 5 and 24 hours post drug treatment. C, A549 (left) and Flo1 (right) cells were fixed and labeled with anti-YAP antibody after 24 hours of drug treatment. Scale bar, 50 μm. D, Quantification of nuclear and cytoplasmic anti-YAP labeling. Relative fluorescence intensity values of anti-YAP labeling in the nuclear (left) and cytoplasmic (right) cellular compartments were normalized to DMSO values and displayed as means ± SEM (n = 3 independent experiments). E, Lysates from Flo1 (top) and A549 (bottom) cells treated as indicated were immunoblotted for Axl and GAPDH. Quantification of Axl expression is displayed as means ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01 (one-way ANOVA).
- Figure 5.
IHC analysis of YAP localization in tumors exposed to a FAK and HDAC inhibitor combination. A, YAP localization was analyzed by IHC in SCC (left), Flo1 (middle), and A549 (right) tumors. Tumor sections were labeled with anti-YAP antibody. Scale bar for 10×, 100 μm, and 40×, 25 μm. B, Quantification of YAP nuclear/cytoplasmic expression at tumor margins. Each symbol represents the average value per tumor, and horizontal black lines represent group means ± SEM [n ≥ 4 mice/group (2 tumors/mouse)]. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA).
Additional Files
Supplementary Data
- Supplementary Fig. S1 - Proteomic analysis of FAK protein complexes and turnover.
- Supplementary Fig. S2 - Analysis of HDAC and FAK inhibition in SCC cells.
- Supplementary Fig. S3 - Analysis of HDAC and FAK inhibition across a panel of 35 human cell lines.
- Supplementary Fig. S4 - Analysis of HDAC and FAK inhibition in A549 and Flo1 cell lines.
- Supplementary Fig. S5 - Quantification of YAP localization in esophageal cell lines.
- Supplementary Table S1 - FAK kinase-dependent dysregulation of specific FAK-binding proteins.
- Supplementary Table S2 - FAK combination phenotypic screen data.
- Supplementary Text - Supplementary Materials and Methods.
Volume 19, Issue 2
