Primary and acquired resistance to conventional chemotherapy and radiotherapy represent the central therapeutic challenge in oncology today. Resistance may develop through varied mechanisms, including increased expression of cellular drug efflux pumps; mutation of the therapeutic target; increased activity of DNA repair mechanisms and altered expression of genes involved in apoptotic pathways. To overcome these resistance mechanisms, conventional cancer treatments are increasingly combined with molecularly targeted therapies. Because cytotoxic and targeted therapies have distinct biologic effects and toxicity profiles, such combinations are both rational and well tolerated. To date, the molecular pathway most frequently targeted in combination with conventional chemotherapy or radiotherapy is that of the EGFR. After activation by binding of the EGF and other natural ligands, EGFR activates prosurvival, pro-angiogenic, and anti-apoptotic pathways that may confer resistance to cytotoxic therapies. Interestingly, all these aforementioned functional pathways are known to be controlled by transcriptional master switch regulator, NFκB that also happens to be a downstream target for EGFR. In this study, we investigated the specific inhibitory effect of EGFR TK inhibitor EKB-569 on the regulation of NFκB-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 NFκB 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 NFκB 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 
. One major downstream signaling route is via Ras-Raf-MAPK pathway 
where activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of ERK1 and 2 
that regulate transcription of molecules that are linked to cell proliferation, survival, and transformation 
. Another important target in EGFR signaling is PI3K and the downstream protein-serine/threonine kinase Akt 
which transduces signals that trigger a cascade of responses from cell growth and proliferation to survival and motility 
. 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 NFκB that mediate cellular responses, including cell division, survival (or death), motility, invasion, adhesion, and cellular repair 
. QPCR profiling revealed a significant increase in these EGFR dependent NFκB activating molecules viz. Akt1
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 
. Transcriptional regulators that regulate 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 pathways leads to the activation of NFκB which in turn imbalance the pro/anti-apoptotic protein expression. As is evident from our data, IR-induced NFκB and NFκB-dependent metabolic activity, cell viability and cell death indicate NFκB's direct role in induced radioresistance. Consistently, in multiple tumor cells, we and others have extensively documented that RT induces NFκB activity and delineated its direct role in induced radioresistance 
. Conversely, muting NFκB function has been shown to restore apoptosis 
and confer apoptotic effect in chemo and/or radioresistant tumor cells 
. Consistently, we observed a complete inhibition of IR-induced NFκB 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 NFκB 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.
It is also interesting to note form this study that the inhibition of NFκB signaling pathway is not a EKB-569 compound-specific effect. Other commonly used irreversible EGFR blockers, afatinib and neratinib (HKI-272) dose-dependently inhibit NFκB DNA-binding activity. The inhibition of NFκB 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 NFκB activity by blocking the activity of IR-induced upstream IκB kinase beta (IKK-β). 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 NFκB DNA-binding activity and mRNA expression in the irradiated cells. Therefore the proposed combination of IR and EGFR/NFκB 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 NFκB signaling pathway transcriptional response. Interestingly, EKB-569 robustly modulates the transcriptional response of NFκB 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 NFκB has recently become an important task in the therapy of several chemoresistant and radioresistant cancers 
. Anti-apoptotic molecules are expressed at high levels in many tumors and have been reported to contribute to the resistance of cancers to RT 
. Because activation of caspases plays a central role in the apoptotic machinery 
, therapeutic modulation of molecules such as IAPs could target the core control point that overturn the cell fate and determine sensitivity to RT 
. A recent body of evidence has emphasized a central role for NFκB in the control of cell proliferation and survival. NFκB 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 
. In this perspective, we recently demonstrated that muting IR-induced NFκB regulates NFκB 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 NFκB 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 radiosensitizer” and further allows us to identify the role of EGFR dependent NFκB 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 machinery, 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 NFκB and subsequent tumor progression. In this regard, p65 subunit of NFκB is constitutively activated in 70% of HNSCC and IR-induced NFκB plays an important role in HNSCC resistance to RT. Though constitutive and RT-induced NFκB 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 present study exhibit that ecotopically muting IR-induced NFκB with ΔIκBα robustly induced cell death in HNSCC cells demonstrating that IR-induced NFκB regulates cell death at least in this setting. Furthermore, to causally delineate that EKB-569 dependent silencing of NFκB mediates the induced radiosensitization, we analyzed their effect on NFκB overexpressed cells. For the first time, the results of the present study imply that EKB-569 inhibits HNSCC cell survival and viability by selectively targeting NFκB.
In summary, these results demonstrate that EKB-569 significantly inhibits IR-induced NFκB activity in human HNSCC cells. Furthermore, this study identifies the EKB-569-associated inhibition of NFκB pathway survival signaling blue print, more precisely to the regimen of the treatment modality, in this case IR. Evidently, treatment with EKB-569 profoundly conferred IR-inhibited HNSCC cell survival and viability. Consistently, this EGFR TK significantly enhanced IR-induced HNSCC apoptosis. More importantly, NFκB over expression and knockout studies demonstrated that EKB-569-associated targeting of IR-induced NFκB mediates cell death in HNSCC cells. Taken together, these data strongly suggest that EKB-569 may exert radiosensitization at least in part by selectively targeting IR-induced NFκB dependent survival signaling, that potentiate radiotherapy in effective HNSCC cell killing. Further in-depth in vivo studies are warranted to verify this suggestion and are presently under investigation in our laboratory.