Focal Adhesion Kinase Drives Resistance to Therapy in HPV-Negative Head and Neck Squamous Cell Carcinoma in a p53-Dependent Manner

Abstract

<h3>Purpose/Objective(s)</h3> Focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative [HPV (-)] head and neck squamous cell carcinoma (HNSCC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but TP53 is mutated in over 80% of HPV (-) HNSCC. Based on these observations, we tested the hypothesis that mutant (mut) TP53 permits or potentially drives FAK-mediated therapy resistance. <h3>Materials/Methods</h3> <i>In vivo</i> shRNA screening in 3 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins combined with RT or carboplatin. An <i>in vivo</i> immunodeficient flank model using HN31 (mut TP53) cells expressing FAK shRNA was used to assess <i>in vivo</i> radioresponse. The relationship between p53, FAK, and radioresponse was examined <i>in vitro</i> using HNSCC cells: UMSCC (p53 null) with wt or mut TP53 expressed, HN30 (wt TP53), and HN31 (mut TP53). FAK function was modulated in these cells using siRNA, shRNA, or chemical inhibition (defactinib); response to radiation (RT) or carboplatin was evaluated via immunoblot for DNA damage repair proteins, survival clonogenic assay, MTT assay, DCFDA reactive oxygen species (ROS) assay, and fluorescence-based homologous recombination (HR) and non-homologous end joining (NHEJ) reporter assays. FAK and p53 interactions were evaluated via immunoprecipitation. Two cohorts of HPV (-) HNSCC tumors treated with RT, single institution (n=81) and TCGA (n=324), were stratified by TP53 mutational status and FAK copy number/mRNA expression. <h3>Results</h3> <i>In vivo</i> shRNA screening identified FAK as an excellent target for both radio- and chemosensitization. Inhibition of FAK via shRNA in the HN31 (mut TP53) cell line led to radiosensitization in a xenograft flank model. Defactinib treatment led to increased sensitivity to RT or carboplatin in mut TP53 (HN31) but not wt TP53 (HN30) tumor cell lines. In UMSCC1 cells treated with defactinib, mut TP53 expression resulted in significant radiosensitization, but wt TP53 expression did not. Both wt p53 and mut p53 retained the ability to bind FAK. Basal FAK phosphorylation was higher in cells expressing mut p53 compared to wt p53. FAK inhibition combined with radiation led to higher levels of ɣ-H2AX and ROS and decreased HR and NHEJ repair in mut TP53 expressing cells but had minimal effect in wt TP53 cells. High levels of FAK copy number and gene expression were associated with worse disease-free survival in mut TP53 HPV (-) HNSCC, but not wt TP53 tumors in both patient cohorts. <h3>Conclusion</h3> FAK is a potentially important target for therapeutic sensitization, primarily in mutated TP53 HPV (-) HNSCC.