Epidermal Growth Factor Receptor Inhibition Reduces Angiogenesis via Hypoxia-Inducible Factor-1α and Notch1 in Head Neck Squamous Cell Carcinoma

Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.     

The epidermal growth factor receptor modulates DNA double-strand break repair by regulating non-homologous end-joining

In mammalian cells repair of radiation-induced DNA damage appears to be also controlled by the epidermal growth factor receptor (EGFR) with a special impact on DNA double-strand break (DSB) repair. Aim of this study was to demonstrate this interaction between EGFR signalling and DNA DSB repair and to identify the underlying downstream pathways. We especially wanted to know in how far non-homologous end-joining (NHEJ) as the most important DSB repair pathway is involved in this interaction. Overall DSB repair was determined by counting γH2AX foci remaining 24 after irradiation, while NHEJ activity was monitored by using a specially designed repair construct stably integrated into the genome. The overall DSB repair capacity was clearly enhanced when EGFR was activated by its natural ligand EGF and, vice versa, was reduced when EGFR was blocked either by the specific antibody Cetuximab or the tyrosine kinase inhibitor erlotinib, whereby reduction was clearly stronger for erlotinib. There was also a difference in the pathways affected. While erlotinib lead to a block of both, MAPK as well as AKT signalling, Cetuximab only affected MAPK. As demonstrated by specific inhibitors (PD98059, AKTIII) EGFR interacts with DSB repair mostly via MAPK pathway. Also for NHEJ activity, there was a substantial increase, when EGFR was activated by EGF as determined for two different reporter cell lines (A549.EJ and H1299.EJ) and, vice versa, a reduction was seen when EGFR signalling was blocked by Cetuximab or erlotinib. There was, however, no difference for the two inhibitors used. This regulation of NHEJ by EGFR was only blocked when ERK was affected by siRNA but not when AKT was knocked down. These data indicate that EGFR modulates DSB repair by regulating NHEJ via MAPK signalling.     

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.     

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.     

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.     

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.     

CHK2-dependent phosphorylation of BRCA1 in hypoxia

Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.     

Inhibition of NF-κB and DNA double-strand break repair by DMAPT sensitizes non-small-cell lung cancers to X-rays

We investigated the efficacy and mechanism of dimethylaminoparthenolide (DMAPT), an NF-κB inhibitor, to sensitize human lung cancer cells to X-ray killing in vitro and in vivo. We tested whether DMAPT increased the effectiveness of single and fractionated X-ray treatment through inhibition of NF-κB and/or DNA double-strand break (DSB) repair. Treatment with DMAPT decreased plating efficiency, inhibited constitutive and radiation-induced NF-κB binding activity, and enhanced radiation-induced cell killing by dose modification factors of 1.8 and 1.4 in vitro. X-ray fractionation demonstrated that DMAPT inhibited split-dose recovery/repair, and neutral DNA comet assays confirmed that DMAPT altered the fast and slow components of X-ray-induced DNA DSB repair. Knockdown of the NF-κB family member p65 by siRNA increased radiation sensitivity and completely inhibited split-dose recovery in a manner very similar to DMAPT treatment. The data suggest a link between inhibition of NF-κB and inhibition of DSB repair by DMAPT that leads to enhancement of X-ray-induced cell killing in vitro in non-small-cell lung cancer cells. Studies of A549 tumor xenografts in nude mice demonstrated that DMAPT enhanced X-ray-induced tumor growth delay in vivo.     

Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.     

Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase

Ionizing radiation, but not stimulation with epidermal growth factor (EGF), triggers EGF receptor (EGFR) import into the nucleus in a probably karyopherin alpha-linked manner. An increase in nuclear EGFR is also observed after treatment with H2O2, heat, or cisplatin. During, this process, the proteins Ku70/80 and the protein phosphatase 1 are transported into the nucleus. As a consequence, an increase in the nuclear kinase activity of DNA-dependent kinase (DNA-PK) and increased formation of the DNA end-binding protein complexes containing DNA-PK, essential for repair of DNA-strand breaks, occurred. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced activation of DNA-PK, inhibited DNA repair, and increased radiosensitivity of treated cells. Our data implicate a novel function of the EGFR during DNA repair processes.     

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice

Background

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.

Methodology/Principal Findings

Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.

Conclusion/Significance

Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.     

Repair of chromatin damage in glutathione-depleted V-79 cells: comparison of oxic and hypoxic conditions

We have assessed the effects of two radiomodifying conditions, glutathione (GSH) depletion and hypoxia, on the formation and repair of radiation-induced chromatin damage, specifically DNA-protein cross-links (DPC). As measured by a nitrocellulose filter-binding assay, untreated V79 cells contain a low level of DPC (1-1.5% of the cellular DNA). The background level of DPC is elevated in cells treated with L-buthionine sulfoximine (BSO), in hypoxic cells, and in cells treated with BSO and made hypoxic (2.98%, 2.82%, and 7.71%, respectively). The dose response for production of radiation-induced DPC is approximately 6.0% DNA bound per 100 Gy for cells irradiated in air, and the dose response is not significantly different for BSO-treated cells but increases by a factor of about 1.4 for hypoxic cells and 1.7 for BSO-pretreated hypoxic cells. DPC were also assayed by alkaline elution with or without proteinase K treatment. By this analysis, the yield of DPC appears to be elevated in irradiated hypoxic and irradiated GSH-depleted cells. It is not possible to assay for background DPC alone in unirradiated cells by alkaline elution. Cells not exposed to BSO repair 70-80% of the radiation-induced DPC in 4 h. BSO-treated cells are considerably less efficient in repair of DPC. As analyzed by alkaline elution, GSH depletion had little or no effect on the yield of radiation-induced single-strand breaks (SSB) but slowed their repair. The data suggest that depletion of GSH impairs an enzyme system(s) responsible for the turnover of both background and radiation-induced DPC and that hypoxia elevates both the background level of DPC and the ratio of radiation-induced DPC to SSB.     

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen-activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)-induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen-activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time- and dose-dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP-GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5-altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho-extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5-induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth.     

Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.     

Inhibition of DNA-dependent protein kinase induces accelerated senescence in irradiated human cancer cells

DNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair of DNA double-strand breaks (DSB) and is centrally involved in regulating cellular radiosensitivity. Here, we identify DNA-PK as a key therapeutic target for augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor of DNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting in cellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement by BEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G(2)-M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21 expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accompanied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces accelerated senescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but not PI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show that PRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cells comparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibition of DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as an important therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade.     

EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK

Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.     

Cetuximab augments cytotoxicity with poly (adp-ribose) polymerase inhibition in head and neck cancer

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.