this study, we investigated the specific inhibitory effect of EGFR TK inhibitor EKB-569 on the regulation of NFkB-dependent survival advantage and elucidated its influence in potentiating radiotherapy for head and neck cancers. To our knowledge, for the first time, we have demonstrated the specific inhibition of IR- induced NFkB with irreversible EGFR TK inhibitor, EKB-569 and dissected out the functional downstream signaling that orchestrate in promoting radiosensitization at least in head neck cancer. Our results indicate that radiation at clinically relevant doses activated NFkB pathway in SCC-4 cells through the mechanism that interacted with EGFR. To that note, activation of EGFR intrinsic receptor protein TK and tyrosine autophosphorylation results in the activation of a number of key signaling pathways [31]. One major downstream signaling route is via Ras-Raf- MAPK pathway [32] where activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of ERK1 and 2 [33] that regulate transcription of molecules that are linked to cell proliferation, survival, and transformation [33]. Another important target in EGFR signaling is PI3K and the downstream protein-serine/threonine kinase Akt [34,35] which transduces signals that trigger a cascade of responses from cell growth and proliferation to survival and motility [35]. One more route is via the stress-activated protein kinase pathway, involving protein kinase C and Jak/Stat. Interestingly, the activation of these pathways converges into distinct transcriptional program involving NFkB that mediate cellular responses, including cell division, survival (or death), motility, invasion, adhesion, and cellular repair [25]. QPCR profiling revealed a significant increase in these EGFR dependent NFkB activating molecules viz. Akt1, Jun, Map3K1, Raf1 after IR and, EKB-569 treatment resulted in complete suppression of these molecules and serve as the positive controls for the study. Transformed cells have been shown to possess deregulated apoptotic machinery [36]. Transcriptional regulators that regu- late pro-apoptotic and/or activate anti-apoptotic proteins play a key role in switching the therapy associated balance of apoptotic cell death. In this regard, EGFR blockers appear to inhibit tumor cell death via multiple mechanisms. EGFR-mediated signaling via the Ras-Raf-MAPK, PI3-K/Akt or PKC-Jak/STAT path- ways leads to the activation of NFkB which in turn imbalance the pro/anti-apoptotic protein expression. As is evident from our data, IR-induced NFkB and NFkB-dependent metabolic activity, cell viability and cell death indicate NFkB’s direct role in induced radioresistance. Consistently, in multiple tumor cells, we and others have extensively documented that RT induces NFkB activity and delineated its direct role in induced radioresistance [29,37–43]. Conversely, muting NFkB function has been shown to restore apoptosis [44] and confer apoptotic effect in chemo and/or radioresistant tumor cells [45]. Consistently, we observed a complete inhibition of IR-induced NFkB activity with EKB-569 designating that this compound may rectify IR-induced aberrant apoptotic machinery. These results though confirmed that the mechanism of EKB-569-mediated radiosensitization of squamous cell carcinoma is acting specifically through NF-kB pathway, it is interesting to note an induction in the activity of other transcription factors, AP-1 and SP-1. This differential mechanism in the activation of NFkB versus AP-1 and SP-1 may be speculated partly as cell type- and/or stimuli-specific. However, addressing the complete mechanism involved in the induction of IR-induced AP-1 and SP-1 with EKB-569 treatment and its impact on radiosensitization compared to other EGFR-TK inhibitors may help in ascertain the complexity in the combination treatments. EKB Radiosensitizes Squamous Cell Carcinoma It is also interesting to note form this study that the inhibition of NFkB signaling pathway is not a EKB-569 compound-specific effect. Other commonly used irreversible EGFR blockers, afatinib and neratinib (HKI-272) dose-dependently inhibit NFkB DNA- binding activity. The inhibition of NFkB by these two related compounds was found to be persistent up to at least 72 h as seen with EKB-569 treatment. Similarly, all three EGFR inhibitors, EKB-569, afatinib and neratinib directly inhibit NFkB activity by blocking the activity of IR-induced upstream IkB kinase beta (IKK-b). This direct action of inhibition of NF-kB is EGFR- dependent. EGFR-knockdown experiments with a widely used specific EGFR inhibitor, PD153035 confirmed the EGFR- mediated inhibition of NFkB DNA-binding activity and mRNA expression in the irradiated cells. Therefore the proposed combination of IR and EGFR/NFkB inhibition can be carried out on to the clinic with any EGFR inhibitor compounds other than EKB-569. To further substantiate our findings, we analyzed the efficacy of EKB-569 in IR-modulated NFkB signaling pathway transcrip- tional response. Interestingly, EKB-569 robustly modulates the transcriptional response of NFkB signal transduction and downstream mediators of this pathway in SCC-4 cells. To that note, EKB-569 inhibited IR-induced transcription of pro-survival molecules in this setting. Disruption of aberrantly regulated survival signaling mediated by NFkB has recently become an important task in the therapy of several chemoresistant and radioresistant cancers [46]. Anti-apoptotic molecules are expressed at high levels in many tumors and have been reported to contribute to the resistance of cancers to RT [47]. Because activation of caspases plays a central role in the apoptotic machinery [47], therapeutic modulation of molecules such as IAPs could target the core control point that overturn the cell fate and determine sensitivity to RT [48–51]. A recent body of evidence has emphasized a central role for NFkB in the control of cell proliferation and survival. NFkB enhances cell survival by switching on the activation of pro-survival molecules that dampen pro-apoptotic signals and attenuate apoptotic response to anticancer drugs and IR [52,53]. In this perspective, we recently demonstrated that muting IR-induced NFkB regulates NFkB dependent pro-survival molecules and potentiate radiosensitization at least in breast cancer and neuroblastoma models. To our knowledge, the present study for the first time throws light on the efficacy of EKB-569 in regulating IR altered NFkB signal transduction and downstream effector molecules in HNSCC cells. This insight into the comprehensive regulation of IR-induced survival transcription recognizes EKB-569 as ‘‘potential radiosen- sitizer’’ and further allows us to identify the role of EGFR dependent NFkB mediated orchestration of radioresistance at least in HNSCC. Though a plethora of studies dissected out the EGFR downstream signaling (some of them discussed above) and suggested that these signaling converge at transcriptional machin- ery, there remained a paucity of information on the role of specific transcriptional switch in orchestrating EGFR dependent tumor progression. Not only, this study throws light on the molecular blue print that underlies after clinical doses of IR in HNSCC, this study also identifies the potential of the EGFR TK, EKB-569 in selectively targeting IR-induced NFkB and subsequent tumor progression. In this regard, p65 subunit of NFkB is constitutively activated in 70% of HNSCC and IR-induced NFkB plays an important role in HNSCC resistance to RT. Though constitutive and RT-induced NFkB has been causally linked to induced- radioresistance, its precise participation in RT-induced cell death orchestration is poorly understood. In this regard, results of the PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e29705