Irreversible EGFR Inhibitor EKB-569 Targets Low-LET γ-Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma

Department of Otolaryngology, Head and Neck Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
PLoS ONE (Impact Factor: 3.53). 12/2011; 6(12):e29705. DOI: 10.1371/journal.pone.0029705
Source: PubMed

ABSTRACT EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted.

Download full-text


Available from: Natarajan Aravindan, Jul 08, 2014
  • Source
    • "Radiation treatment of bladder cancer cells dose-dependently induced phosphorylation of EGFR, HER2 and Akt; in a colony formation assay, afatinib showed synergism with radiation in such cells, indicating possible radiosensitization (Tasi et al. 2012). A minor radiosensitization by afatinib had also been observed in HNSCC cell lines (Aravindan et al. 2011; Schütze et al. 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Afatinib (also known as BIBW 2992) has recently been approved in several countries for the treatment of a distinct type of epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer. This manuscript comprehensively reviews the preclinical data on afatinib, an irreversible inhibitor of the tyrosine kinase activity of members of the epidermal growth factor receptor family (ErbB) including EGFR, HER2 and ErbB4. Afatinib covalently binds to cysteine 797 of the EGFR and the corresponding cysteines 805 and 803 in HER2 and ErbB4, respectively. Such covalent binding irreversibly inhibits the tyrosine kinase activity of these receptors, resulting in reduced auto- and transphosphorylation within the ErbB dimers and inhibition of important steps in the signal transduction of all ErbB receptor family members. Afatinib inhibits cellular growth and induces apoptosis in a wide range of cells representative for non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell cancer and several other cancer types exhibiting abnormalities of the ErbB network. This translates into tumour shrinkage in a variety of in vivo rodent models of such cancers. Afatinib retains inhibitory effects on signal transduction and in vitro and in vivo cancer cell growth in tumours resistant to reversible EGFR inhibitors, such as those exhibiting the T790M mutations. Several combination treatments have been explored to prevent and/or overcome development of resistance to afatinib, the most promising being those with EGFR- or HER2-targeted antibodies, other tyrosine kinase inhibitors or inhibitors of downstream signalling molecules.
    Archiv für Experimentelle Pathologie und Pharmakologie 03/2014; 387(6). DOI:10.1007/s00210-014-0967-3 · 2.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Our earlier studies indicated that ionizing radiation (IR) induces NF-κB-dependent clonal expansion of therapy resistant tumor cells. Herein, we investigated whether mitigation of NF-κB-dependent telomerase activation by EGFR tyrosine kinase inhibitor can enhance IR-induced celling killing. SCC-4 and SCC-9 cells exposed to IR with or without Pelitinib were examined for NF-κB and hTERT transcription using luciferase reporter assays. NF-κB-dependent hTERT transcription was confirmed by either muting NF-κB or by using hTERT constructs lacking NF-κB binding sites. hTERT, mRNA, telomerase activity and cell survival of tumor cells were analyzed using QPCR, TRAP and clonogenic assay, respectively. Pelitinib inhibited IR-induced NF-κB, telomerase activity and hTERT transactivation. Ionizing radiation-induced telomerase activity is regulated at the transcriptional level by triggering TERT promoter activation. Functional NF-κB mediates telomerase activity by binding to the κB binding region in the promoter region of TERT. Elimination of the NF-κB recognition site on telomerase or muting NF-κB compromises IR-induced telomerase promoter activation. We found that Pelitinib inhibited IR-induced TERT transcription, transactivation and telomerase activation in IR-exposed and NF-κB-overexpressed cells. Furthermore, Pelitinib potentiates IR-induced cell killing. Our results strongly suggest that IR-induced NF-κB-mediated cell survival is supported by telomerase activation. We propose that if this pathway can be inhibited with Pelitinib treatment, one could further enhance therapeutic outcome in squamous cell carcinoma.
    Radiation Research 02/2013; 179(3). DOI:10.1667/RR3028.1 · 2.45 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ascertaining function-specific orchestration of NFκB in response to radiation may reveal a molecular blue-print that dictates induced relapse and metastasis of the neuroblastoma. We recently demonstrated that sustained activation of NFκB caused by ionizing radiation (IR)-initiated TNFα-NFκB feedback signaling leads to radioresistance and recurrence of neuroblastoma. We investigated whether muting IR-triggered or TNFα-dependent second-signaling feedback-dependent NFκB nuclear import results in limiting IR-altered invasion and metastasis. Neuroblastoma cells were exposed to 2 Gy and incubated for 1 h or 24 h. The cells were then treated with an NFκB-targeting peptide blocker, SN50. Upon confirming the blockade in DNA-binding activity, transcription driven transactivation of NFκB and secretion of soluble TNFα, transcriptional alterations of 93 tumor invasion/metastasis genes were assessed by using QPCR profiling and then were selectively validated at the protein level. Exposure to 2 Gy induced 63, 42 and 71 genes in surviving SH-SY5Y, IMR-32 and SK-N-MC cells, respectively. Blocking post-translational nuclear import of NFκB comprehensively inhibited both initial activation of genes (62/63, 34/42 and 65/71) triggered by IR and also TNFα-mediated second signaling-dependent sustained (59/63, 32/42 and 71/71) activation of tumor invasion and metastasis signaling molecules. Furthermore, alterations in the proteins MMP9, MMP2, PYK-2, SPA-1, Dnmt3b, Ask-1, CTGF, MMP10, MTA-2, NF-2, E-Cadherin, TIMP-2 and ADAMTS1 and the results of our scratch-wound assay validate the role of post-translational NFκB in IR-regulated invasion/metastasis. These data demonstrate that IR-induced second-phase (post-translational) NFκB activation mediates TNFα-dependent second signaling and further implies that IR induced NFκB in cells that survive after treatment regulates tumor invasion/metastasis signaling.
    Clinical and Experimental Metastasis 04/2013; DOI:10.1007/s10585-013-9580-y · 3.73 Impact Factor