IGF1R and Src inhibition induce synergistic cytotoxicity in HNSCC through inhibition of FAK

Abstract

Head and neck cancer is the sixth most common cancer worldwide with a 5-year survival of only 65%. Targeting compensatory signaling pathways may improve therapeutic responses and combat resistance. Utilizing reverse phase protein arrays (RPPA) to assess the proteome and explore mechanisms of synergistic growth inhibition in HNSCC cell lines treated with IGF1R and Src inhibitors, BMS754807 and dasatinib, respectively, we identified focal adhesion signaling as a critical node. Focal Adhesion Kinase (FAK) and Paxillin phosphorylation were decreased as early as 15 min after treatment, and treatment with a FAK inhibitor, PF-562,271, was sufficient to decrease viability in vitro. Treatment of 3D spheroids demonstrated robust cytotoxicity suggesting that the combination of BMS754807 and dasatinib is effective in multiple experimental models. Furthermore, treatment with BMS754807 and dasatinib significantly decreased cell motility, migration, and invasion in multiple HNSCC cell lines. Most strikingly, treatment with BMS754807 and dasatinib, or a FAK inhibitor alone, significantly increased cleaved-PARP in human ex-vivo HNSCC patient tissues demonstrating a potential clinical utility for targeting FAK or the combined targeting of the IGF1R with Src. This ex-vivo result further confirms FAK as a vital signaling node of this combinatorial treatment and demonstrates therapeutic potential for targeting FAK in HNSCC patients.

Figure 1

Focal adhesion proteins that are upregulated in HNSCC are synergistically decreased with BMS754807 and dasatinib. Lysates from five HNSCC cell lines were assessed by reverse phase protein array (RPPA) to determine protein changes following treatment with vehicle, each single drug, or the combination at 15 min, 1 h, 3 h, 8 h and 24 h. Synergistic changes are defined as “Log Fold Change (LogFC) in combination—(1/2LogFC Drug 1)—(1/2LogFC Drug 2)” with a false discovery rate (FDR) < 0.05. (A) Representative heat map depicting all synergistic changes among the 145 epitopes assessed by RPPA. (B) Heat map depicting synergistic changes in the focal adhesion proteins Pyk2 Y402, Paxillin Y118 and FAK Y576-577. (C) Graphs depict changes in protein expression from treatment with control, BMS754807, dasatinib or BMS754807 and dasatinib combination. (D) mRNA expression in tumor or normal head and neck tissues from the TCGA panCancer Atlas. The “All Samples” graph depicts the reads per kilobase of transcript per million mapped reads (RPKM) value from 522 tumor samples and 44 normal samples. The “Matched samples” compares the RPKM of 43 patients with both tumor and normal sample.

Figure 2

Treatment with BMS754807 and dasatinib alter focal adhesion signaling. Cal27 (A), SCC25 (B), OSC19 (C), FaDu (D) cells were treated with 1 µM BMS754807 ± 10 nM des(1–3)IGF-1, 25 nM dasatinib, or the combination of 1 µM BMS754807 and 25 nM dasatinib as indicated and immunoblot was performed. Representative blots from 3 to 5 independent experiments are shown. Each section of the membrane was probed independently for the respective labeled antibody and imaged independently when necessary to insure appropriate exposure. Each cell line was probed independently, cropping is delineated by white spacing between epitopes or black vertical line. Uncropped western blot images are shown in Supplemental Fig. 8. Changes in FAK Y397 (E) and paxillin Y118 (F) are quantified relative to untreated and represent the mean and SEM of 3–5 independent experiments. (G) SCC25 cells treated with 250 nM linsitinib or 25 nM dasatinib singly or in combination as in (A–E) and (H) quantified relative to untreated and represent the mean and SEM of 3 independent experiments. Asterisks represent p < 0.05 as determined by one-way ANOVA and Tukey post-hoc analysis.

Figure 3

Treatment with BMS754807 and dasatinib decreases viability through a FAK dependent mechanism. Indicated cell lines were treated with varying concentrations of PF-562,271 (A) or defactinib (B) for 72 h in 2D culture followed by quantification using CyQUANT. (C,D) cell lines were treated with varying concentrations of PF-562,271 (C) or defactinib (D) for 9 days in a clonogenic assay followed by quantification crystal violet staining. Cal27 (E), SCC25 (F), OSC19 (G), and FaDu (H) cells were treated with PF-562,271 (PF) alone or in combination with BMS754807 (BMS) and dasatinib (Dasat) followed by CyQUANT assay to determine relative cell number. Data represents the mean ± SEM of 3 independent replicates for each cell line. Asterisks represent p < 0.05 as determined by one-way ANOVA and Tukey post-hoc analysis.

Figure 4

Treatment with BMS754807 and dasatinib decreases motility, migration and invasion of HNSCC. (A) Graphs represent mean wound closure ± SEM in each cell line. (B) Graphs represent relative migration of cells through a Boyden chamber as compared to untreated cells. (C) Relative invasion of HNSCC through a Matrigel-coated Boyden chamber as compared to untreated cells. Quantification by ImageJ. Data is depicted by representation for each independent replicate and the mean ± SEM of at least 5 independent replicates. Asterisks represent p < 0.05 as determined by one-way ANOVA and Tukey post-hoc analysis.

Figure 5

Inhibition of FAK, IGF1R and Src induces cytotoxicity in HNSCC spheroids. (A–C) Efficacy of BMS754807 and dasatinib on Cal27, Osc19, and FaDu on tumor spheroids following 7 days treatment. Data is presented as the mean ± SEM of 3–4 independent biological replicates. (D) PF-562,271 dose response of tumor spheroids. Asterisks represent p < 0.05 as determined by one-way ANOVA and Tukey post-hoc analysis.

Figure 6

Treatment of ex vivo HNSCC tumor tissue with PF-562,271 or BMS754807 and dasatinib induces significant apoptosis. (A) Representation of the work-flow used to analyze human HNSCC tumor ex vivo tissue samples. The image was created with BioRender.com. (B) Representative histology images stained as indicated. (C) Graph represents the fold change in apoptosis as determined by cleaved-PARP staining in each of 6 ex vivo HNSCC tissue samples treated with either BMS754807, dasatinib, BMS754807 and dasatinib or PF-562,271.