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Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC). Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)–negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients. Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy. Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600–7. ©2017 AACR.

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach. Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis. Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively). Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

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.

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC). Experimental Design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data. Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo. Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted. Clin Cancer Res; 17(7); 1815–27. ©2011 AACR.

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years. Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.

Proton radiotherapy (PRT) has shown to be less toxic in the treatment of head and neck cancers (HNSCC) than photon radiotherapy (XRT). XRT causes multiple types of cancer cell death by inducing DNA double-strand breaks. The DNA damage induced by PRT is more complex and has more severe biological consequences versus XRT. The present study was undertaken to uncover the cell death caused by PRT versus XRT in human papillomavirus positive (HPV+, frequently p53 wild-type, p16 positive; p53, p16 are key factors for cell death) and HPV-negative (HPV-, frequently p53 mutant, p16 negative) HNSCC cells. HNSCC cell lines HN5, SqCC/Y1 (HPV-) and UMSCC-47, UPCI-SCC-154 (HPV+) were used. Single doses of 4 Gy irradiation induced cell apoptosis, necrosis, mitotic catastrophe, and senescence were determined. Clinical 200-MeV proton beams (18 cm × 18 cm field) or 6-MV X-ray beams (25 cm × 25 cm field) were used. Cells were positioned in the centers of the irradiation fields (in the middle of the spread-out Bragg peak for PRT). At 4 hours (h), 24h and 48h after PRT or XRT, cell necrosis and apoptosis were determined by Annexin VFITC-conjugated staining. XRT and PRT caused increased cell necrosis in the HPV– SqCC/Y1 (moderate at 24h, significant at 48h [p all < 0.05]) and HN5 (moderate at 24h, 48h) cells, at slightly higher rates in PRT versus XRT treated cells; and in the HPV+ UPCI-SCC-154 (moderate at 4h) and UMSCC-47 (moderate at 48h) cells, with no difference between PRT versus XRT treatment groups. XRT and PRT induced a small percentage of cell apoptosis (range: 1.1 ± 0.5 to 7.6 ± 2.9). Cell apoptosis was only moderately increased in the HPV+ UMSCC-47 (at 4h, 24h, 48h) and HPV– SqCC/Y1 (at 24h, 48h) cells, with slightly more apoptosis seen in the XRT versus the PRT group. Cell senescence was assessed using a well-established senescence-associated biomarker (SA-β-gal). XRT and PRT induced significantly increased cellular senescence at both 4 and 6 days in all four cell lines (p all < 0.01), with PRT treatment leading to a significantly higher percentage of senescence in cells than XRT (p all < 0.01). Mitotic catastrophe, an important cell death mechanism by which solid tumors respond to clinical radiotherapy, was determined using co-staining for cytoplasm with γ- tubulin (with Texas red) and for nucleus with Dapi (blue). XRT and PRT both led to a significantly increased number of cells undergoing mitotic catastrophe at 4h, 24h, 48h and 72h (p all < 0.05) in all four cell lines, with significantly higher rates seen in PRT treated versus XRT treated (p all < 0.05). Moreover, the peak of cell mitotic catastrophe induced by PRT occurred at 48h in the HPV– cells, versus at 72h in the HPV+ cells; while no obvious peak was observed in the XRT treated group. In conclusion, mitotic catastrophe and senescence are the major types of cell death induced by both XRT and PRT, in which PRT inducing higher levels of both types of cell death as compared to XRT. Citation Format: Li Wang, Shichao Han, Jinming Zhu, Xiaochun Wang, Yuting Li, Zeming Wang, Eric Lin, Xiaofang Wang, David P. Molkentine, Pierre Blanchard, Yining Yang, Ruiping Zhang, Narayan Sahoo, Michael Gillin, Xiaorong Ronald Zhu, Xiaodong Zhang, Jeffrey N. Myers, Steven J. Frank. Proton versus photon irradiation induced cell death in head and neck cancer cells with different human papillomavirus status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 69.