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

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

Novel synthetic monoketone transmute radiation-triggered NFκB-dependent TNFα cross-signaling feedback maintained NFκB and favors neuroblastoma regression.

Recently, we demonstrated that radiation (IR) instigates the occurrence of a NFκB-TNFα feedback cycle which sustains persistent NFκB activation in neuroblastoma (NB) cells and favors survival advantage and clonal expansion. Further, we reported that curcumin targets IR-induced survival signaling and NFκB dependent hTERT mediated clonal expansion in human NB cells. Herein, we investigated the efficacy of a novel synthetic monoketone, EF24, a curcumin analog in inhibiting persistent NFκB activation by disrupting the IR-induced NFκB-TNFα-NFκB feedback signaling in NB and subsequent mitigation of survival advantage and clonal expansion. EF24 profoundly suppressed the IR-induced NFκB-DNA binding activity/promoter activation and, maintained the NFκB repression by deterring NFκB-dependent TNFα transactivation/intercellular secretion in genetically varied human NB (SH-SY5Y, IMR-32, SK-PN-DW, MC-IXC and SK-N-MC) cell types. Further, EF24 completely suppressed IR-induced NFκB-TNFα cross-signaling dependent transactivation/translation of pro-survival IAP1, IAP2 and Survivin and subsequent cell survival. In corroboration, EF24 treatment maximally blocked IR-induced NFκB dependent hTERT transactivation/promoter activation, telomerase activation and consequent clonal expansion. EF24 displayed significant regulation of IR-induced feedback dependent NFκB and NFκB mediated survival signaling and complete regression of NB xenograft. Together, the results demonstrate for the first time that, novel synthetic monoketone EF24 potentiates radiotherapy and mitigates NB progression by selectively targeting IR-triggered NFκB-dependent TNFα-NFκB cross-signaling maintained NFκB mediated survival advantage and clonal expansion

MicroRNAs in neuroblastoma tumorigenesis, therapy resistance, and disease evolution

Neuroblastoma (NB) deriving from neural crest cells is the most common extra-cranial solid cancer at infancy. NB originates within the peripheral sympathetic ganglia in adrenal medulla and along the midline of the body. Clinically, NB exhibits significant heterogeneity stretching from spontaneous regression to rapid progression to therapy resistance. MicroRNAs (miRNAs, miRs) are small (19-22 nt in length) non-coding RNAs that regulate human gene expression at the post-transcriptional level and are known to regulate cellular signaling, growth, differentiation, death, stemness, and maintenance. Consequently, the function of miRs in tumorigenesis, progression and resistance is of utmost importance for the understanding of dysfunctional cellular pathways that lead to disease evolution, therapy resistance, and poor clinical outcomes. Over the last two decades, much attention has been devoted to understanding the functional roles of miRs in NB biology. This review focuses on highlighting the important implications of miRs within the context of NB disease progression, particularly miRs’ influences on NB disease evolution and therapy resistance. In this review, we discuss the functions of both the “oncomiRs” and “tumor suppressor miRs” in NB progression/therapy resistance. These are the critical components to be considered during the development of novel miR-based therapeutic strategies to counter therapy resistance

Cancer stem cells in neuroblastoma therapy resistance

Neuroblastoma (NB) is the most common cancer of infancy and accounts for nearly one tenth of pediatric cancer deaths. This mortality rate has been attributed to the > 50% frequency of relapse despite intensive, multimodal clinical therapy in patients with progressive NB. Given the disease’s heterogeneity and developed resistance, attaining a cure after relapse of progressive NB is highly challenging. A rapid decrease in the timeline between successive recurrences is likely due to the ongoing acquisition of genetic rearrangements in undifferentiated NB-cancer stem cells (CSCs). In this review, we present the current understanding of NB-CSCs, their intrinsic role in tumorigenesis, their function in disease progression, and their influence on acquired therapy resistance and tumor evolution. In particular, this review focus on the intrinsic involvement of stem cells and signaling in the genesis of NB, the function of pre-existing CSCs in NB progression and therapy response, the formation and influence of induced CSCs (iCSCs) in drug resistance and tumor evolution, and the development of a CSC-targeted therapeutic approach

EF24 regulates radiation-induced NFκB downstream pro-survival proteins in human neuroblastoma cells.

<p>(<b>A</b>) Immunoblots showing alterations in pIκBα, IAP1, IAP2 and Survivin protein expression in SH-SY5Y, IMR-32, SK–PN–DW and MC-IXC cells either mock-irradiated, exposed to 2Gy and harvested after 24h, treated with EF24 for 3h followed by 2Gy exposure and harvested after 1, 3, 12, 24, 48 and 72h, transfected with p50/p65 for 24h or transfected with p50/p65 for 24h and treated with EF24 for additional 24h. (<b>B</b>) Semi-quantitative densitometry of immunoblots using Quantity One 1D image analysis Version 4.6.5 (Biorad) showing α-tubulin intensity normalized expression of IAP1, IAP2 and Survivin in SH-SY5Y, IMR-32, SK–PN–DW and MC-IXC cells. Groups were compared using Two-way ANOVA with Bonferroni’s Post-hoc correction.</p

EF24 regulates radiation-induced telomerase activation in human neuroblastoma cells.

<p>Representative gels and corresponding densitometry analysis showing telomerase activity in (<b>A</b>) MC-IXC, (<b>B</b>) SH-SY5Y, (<b>C</b>) SK–PN–DW and (<b>D</b>) IMR-32 cells either mock-irradiated; exposed to 2Gy and harvested after 6, 24, 48 and 72h; treated with 10, 50, 100 and 200nM EF24 for 3h followed by IR exposure and harvested after 3h, treated with 200nM EF24 for 34 followed by IR exposure and harvested after 1, 3, 6, 12, 24, 48, 72 and 96h; transfected with p50/p65 for 24h or transfected with p50/p65 for 24h and treated with EF24 for additional 24h. Densitometry analysis with automatic band detection (ImageQuant TL, Amersham Biosciences) showed significant inhibition of either 2Gy- or p50/p65-induced telomerase activity with EF24.</p

EF24 inhibits radiation-induced NFκB dependent initiation of clonal expansion in human neuroblastoma cells.

<p>(<b>A</b>) Representative images showing clonogenic activity of SH-SY5Y and IMR-32 cells either mock irradiated, treated with increasing (50, 100 and 200nM) concentrations of EF24 or exposed to 2Gy with or without EF24 (200nM) treatment, transfected with p50/p65 with or without EF24 treatment. The colonies were fixed, stained with 0.5% crystal violet, and imaged. (<b>B</b>) Histograms showing dose dependent inhibition of clonal expansion in Ef24 treated SH-SY5Y and IMR-32 cells. Histograms showing clonogenic capacity of (<b>C</b>) SH-SY5Y and (<b>D</b>) IMR-32 cells exposed to IR, treated with EF24 and exposed to IR, transfected with p50/p65 and treated with or without EF24. The colonies were counted using computed colony counting (Image Quant). Groups were compared using ANOVA with Tukey’s Post-hoc correction.</p