David P. Molkentine

DNA repair biomarker profiling of head and neck cancer: Ku80 expression predicts locoregional failure and death following radiotherapy

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.

Vandetanib Restores Head and Neck Squamous Cell Carcinoma Cells' Sensitivity to Cisplatin and Radiation In Vivo and In Vitro

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.

Dasatinib, a multi-kinase inhibitor increased radiation sensitivity by interfering with nuclear localization of epidermal growth factor receptor and by blocking DNA repair pathways

BACKGROUND AND PURPOSE Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect. MATERIALS AND METHODS Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells. RESULTS All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity. CONCLUSIONS Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.

A novel Chk inhibitor, XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe.

SummaryCheck point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.

Combination of Anti-IGF-1R Antibody A12 and Ionizing Radiation in Upper Respiratory Tract Cancers

PURPOSE The IGF1/IGF-1R signaling pathway has emerged as a potential determinant of radiation resistance in human cancer cell lines. Therefore we investigated the potency of monoclonal anti-IGF-1R antibody, A12, to enhance radiation response in upper respiratory tract cancers. METHODS AND MATERIALS Cell lines were assessed for IGF-1R expression and IGF1-dependent response to A12 or radiation using viability and clonogenic cancer cell survival assays. In vivo response of tumor xenografts to 10 or 20 Gy and A12 (0.25-2 mg × 3) was assessed using growth delay assays. Combined treatment effects were also analyzed by immunohistochemical assays for tumor cell proliferation, apoptosis, necrosis, and vascular endothelial growth factor expression at Days 1 and 6 after start of treatment. RESULTS A12 enhanced the radiosensitivity of HN5 and FaDu head-and-neck carcinomas in vitro (p < 0.05) and amplified the radioresponse of FaDu xenografts in a dose-dependent manner, with enhancement factors ranging from 1.2 to 1.8 (p < 0.01). Immunohistochemical analysis of FaDu xenografts demonstrated that A12 inhibited tumor cell proliferation (p < 0.05) and vascular endothelial growth factor expression. When A12 was combined with radiation, this resulted in apoptosis induction that persisted until 6 days from the start of treatment and in increased necrosis at Day 1 (p < 0.01, respectively). Combined treatment with A12 and radiation resulted in additive or subadditive growth delay in H460 or A549 xenografts, respectively. CONCLUSIONS The results of this study strengthen the evidence for investigating how anti-IGF-1R strategies can be integrated into radiation and radiation-cetuximab regimen in the treatment of cancer of the upper aerodigestive tract cancers.

C-Met Inhibitor MK-8003 Radiosensitizes c-Met–Expressing Non–Small-Cell Lung Cancer Cells With Radiation-Induced c-Met–Expression

Introduction: The radiation doses used to treat unresectable lung cancer are often limited by the proximity of normal tissues. Overexpression of c-Met, a receptor tyrosine kinase, occurs in about half of non–small-cell lung cancers (NSCLCs) and has been associated with resistance to radiation therapy and poor patient survival. We hypothesized that inhibiting c-Met would increase the sensitivity of NSCLC cells to radiation, enhancing the therapeutic ratio, which may potentially translate into improved local control. Methods: We tested the radiosensitivity of two high-c-Met–expressing NSCLC lines, EBC-1 and H1993, and two low-c-Met–expressing lines, A549 and H460, with and without the small-molecule c-Met inhibitor MK-8033. Proliferation and protein expression were measured with clonogenic survival assays and Western blotting, respectively. &ggr;-H2AX levels were evaluated by immunofluorescence staining. Results: MK-8033 radiosensitized the high-c-Met–expressing EBC-1 and H1993 cells but not the low-c-Met–expressing cell lines A549 and H460. However, irradiation of A549 and H460 cells increased the expression of c-Met protein at 30 minutes after the irradiation. Subsequent targeting of this up-regulated c-Met by using MK-8033 followed by a second radiation dose reduced the clonogenic survival of both A549 and H460 cells. MK-8033 reduced the levels of radiation-induced phosphorylated (activated) c-Met in A549 cells. Conclusions: These results suggest that inhibition of c-Met could be an effective strategy to radiosensitize NSCLC tumors with high basal c-Met expression or tumors that acquired resistance to radiation because of up-regulation of c-Met.

Proteomic profiling identifies PTK2/FAK as a driver of radioresistance in HPV-negative head and neck cancer

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.

Integrative Analysis Identifies a Novel AXL–PI3 Kinase–PD-L1 Signaling Axis Associated with Radiation Resistance in Head and Neck Cancer

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.

TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects.

Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. p16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. p16-overexpressing and small hairpin RNA-knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction; and data from The Cancer Genome Atlas HNSCC project were analyzed. p16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.

Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit

Interaction between the epidermal growth factor receptor (EGFR) and the insulin‐like growth factor receptor (IGF‐1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC‐A12 (IGF‐1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC‐A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ‐H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC‐A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ‐H2AX and 53BP1 foci. IMC‐A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF‐1R expression levels and also the association between EGFR and IGF‐1R. Addition of IMC‐A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC‐A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF‐1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.