Proteomic Profiling Identifies PTK2/FAK as a Driver of Radioresistance in HPV-negative Head and Neck Cancer

Abstract

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC.

Experimental design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC.

Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2-M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas.

Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643-50. ©2016 AACR.

Figure 1. Proteomic profiling identifies markers of radioresistance in HPV-negative HNSCC cell lines

(A) Reverse phase protein array analysis of 49 HNSCC cell lines, illustrated as unsupervised hierarchical clustering. Shown are proteins and phospho-proteins differentially expressed between radioresistant (red bars) and radiosensitive (blue bars) cell lines (false discovery rate 1%, mean fold difference ≥ 1.5). (B) Graphical representation of proteins and phospho-proteins differentially expressed between groups.

Figure 2. PTK2/FAK mRNA expression is significantly higher in radioresistant HPV-negative HNSCC

(A) Genes encoding proteins expressed differently in the RPPA analysis (Fig. 1) were examined similarly, comparing radioresistant (red bars) and radiosensitive (blue bar) groups in hierarchical clustering. (B) PTK2/FAK gene expression was significantly higher in radioresistant HNSCC cell lines.

Figure 3. Inhibition of PTK2/FAK, but not FGFR, leads to radiosensitization in HPV negative HNSCC cells

(A) The FGFR inhibitor nintedanib does not radiosensitize HN5 HNSCC cells. B & C) (B) The FAK inhibitor PF562271 radiosensitizes HN5 HNSCC cells and other HNSCC cell lines (C). P values for each radiation dose comparison are shown.

Figure 4. PTK2/FAK copy number is highly associated with mRNA expression and protein level in HPV-negative HNSCC

(A) Both PTK2/FAK gene expression (Pearson r=0.55, P=0.001) and copy number (Pearson r=0.46, P=0.008) are significantly correlated with protein level in the tested HNSCC cell lines. (B) PTK2/FAK copy number is significantly correlated with gene expression in samples from The Cancer Genome Atlas patients with known HPV-negative head and neck cancer (n=243; Pearson r=0.51, P<0.0001).

Figure 5. PTK2/FAK copy number is associated with disease recurrence in HPV-negative HNSCC

PTK2/FAK copy number is associated with poorer disease-free survival (DFS) in an inpatients with HNSCC treated with radiotherapy at MD Anderson, considered both separately (A and B) and combined (C). PTK2/FAK copy number trends to association with DFS in the Head and Neck Cancer TCGA cohort as well (P=0.063).

Figure 6. PTK2/FAK mRNA expression is associated with disease recurrence in patients with HPV-negative HNSCC

PTK2/FAK mRNA expression is associated with DFS in an institutional cohort of 102 patients with HPV-negative HNSCC (A) and in patients from the Head and Neck Cancer TCGA cohort (B). (C) Kaplan Meier curve showing DFS in patients from the TCGA cohort split at median PTK2/FAK expression.