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

Abstract

Our group has previously demonstrated focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative (HPV-) head and neck squamous cell carcinoma (HNC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but p53 is mutated in over 80% of HPV(-) HNC. Based on these observations, we tested the hypothesis that mutant (mut) p53 permits or even drives FAK-mediated radioresistance.Two cohorts of HPV(-) HNC tumors treated with radiation (RT), single institution (n = 81) and TCGA (n = 324), were stratified by TP53 mutational status (wt versus mut) and FAK copy number/mRNA expression. In vivo shRNA screening in 5 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins and DNA damage repair combined with RT or carboplatin. Correlation between FAK protein expression and sensitivity to cisplatin was evaluated in 20 TP53 mut HNC cell lines. The relationship between p53, FAK and radioresponse was examined in vitro using UMSCC (p53 null) HNC cells forced to express wt or mut TP53. FAK function was modulated in these cells using shRNA or chemical inhibition (defactinib) and response to radiation or carboplatin was evaluated via immunoblot for DNA damage repair proteins and clonogenic assay. FAK and p53 interactions were evaluated via immunoprecipitation. An in vivo immunodeficient flank model using HN31 cells expressing FAK shRNA was used to assess in vivo radioresponse.High levels of FAK copy number and gene expression were associated with worse disease-free survival in TP53 mut HPV(-) HNC, but not TP53 wt tumors. In vivo shRNA screening identified FAK as an excellent target for either radio- or chemosensitization. FAK protein expression was correlated with cisplatin IC50 (P = 0.0083, r = 0.572) in our HNC cell line panel. Inhibition of FAK via shRNA in the HN31(TP53 mut) cell line led to radiosensitization both in vitro and in a xenograft flank model. Defactinib treatment led to increased sensitivity to radiation or carboplatin in TP53 mut (HN31) but not TP53 wt (HN30) tumor cell lines. In UMSCC1 cells pretreated with defactinib, TP53 mut expression resulted in significant radiosensitization, but TP53 wt expression did not. To examine the physical interaction between p53 and FAK, different TP53 constructs were expressed in UMSCC1 cells. Interestingly, missense mutation does not impair the ability of p53 to bind to FAK. However, FAK phosphorylation was higher in cells expressing mut p53 compared to wt. Moreover, FAK inhibition combined with radiation led to higher levels of ɣ-H2AX in mut p53 expressing cells.FAK is a potentially important target for therapeutic sensitization, primarily in TP53 mutated HNSCC.