The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

Abstract

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53–p21^Cip1/Waf1 axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H₂O₂. The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H₂O₂-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53–p21^Cip1/Waf1 axis; but inhibiting the canonical NF-κB pathway exacerbated H₂O₂-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H₂O₂-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.

Introduction

Oxidative DNA damage results from the accumulation of reactive oxygen species (ROS) due to an imbalance between ROS production and removal by antioxidant systems. ROS cause a variety of genetic damage, mostly base lesions and DNA single strand breaks (SSBs) but also double stranded DNA breaks (DSBs). In effect, replication can stall due to DNA damage resulting from ROS in the cell, and failure to stabilize stalled replication forks can result in their collapse and ultimately genetic instability [1], [2]. Oxidative damage can provoke cell cycle arrest and senescence, but it can also induce apoptosis with normal cells being more resistant to apoptosis than tumor cells [1], [2], [3].

Normal human diploid fibroblasts (HDFs) undergo a finite number of cell divisions in culture, a phenomenon termed cellular or replicative senescence. In vitro cell propagation causes telomere shortening, recognized as DSBs that activates a DNA damage checkpoint response (DDR), culminating in replicative senescence. In addition, human cells undergoing senescence and aging mice accumulate DNA lesions with irreparable DSBs outside telomeres, suggesting that their accumulation may have a causal role in mammalian aging [4], [5], [6]. DDR involves activation of the kinases ATM and Chk2 and their downstream effector p53 and its target p21^Cip1/Waf1 [7], [8]. Normal HDFs can also undergo senescence in response to oxidative stress, referred to as stress-induced premature senescence (SIPS) [8], [9]. Hydrogen peroxide (H₂O₂) produces thymidine glycols that can lead to DSBs at replication forks [1] and to the appearance of DNA damage foci in HDFs [4], [9], [10]. ROS accumulation was shown to accelerate HDF senescence by DDR in conjunction with the induction and stabilization of p53 and p21^Cip1/Waf1 [11], [12], [13], [14], [15], [16]. However, dependent on cell type and dosage, H₂O₂-mediated ROS accumulation has also been reported to induce the death of normal and tumor cells [1], [3], [17], [18].

NF-κB transcription factors are critical regulators of most if not all pro-inflammatory/stress-like responses. NF-κBs bind to DNA as dimers of five possible subunits (RelA/p65, c-Rel, RelB, p50, p52). Archetypical p65/p50 heterodimers are cytoplasmically restrained by IκBs (inhibitors of NF-κBs) in most cells. Canonical NF-κB activation generally requires the phosphorylation of serines 32 and 36 in ΙκΒα's signal response domain (SRD), causing its ubiquitination and subsequent proteasomal destruction, allowing p65/50 dimers to translocate to the nucleus and activate their target genes. IκBα SRD phosphorylation requires the IKK signalsome complex (IKKα and IKKβ serine–threonine kinases, and NEMO/IKKγ, a regulatory/adapter protein). IKKβ activation by phosphorylation of its T-activating loop serines 177/181 requires NEMO and rapidly occurs in response to a host of pro-inflammatory/stress-related extracellular signals. In contrast to IKKβ, IKKα activation, by phosphorylation of T-loop serines 176/180, is NEMO-independent, requires de novo protein synthesis and is mainly mediated by stimuli invoked in adaptive immune responses. In vivo, unlike IKKβ, IKKα does not generally target IκBs but instead phosphorylates an IκB-like signal SRD in NF-κB p100, which induces its ubiquitination/proteasome-dependent processing yielding the NF-κB p52 subunit. Thus, NF-κB p100 functions akin to IκBα by sequestering NF-κB RelB in the cytoplasm and also yields NF-κB p52, which translocates to the nucleus together with RelB, to activate other unique NF-κB target genes [19], [20]. Some p100 also sequesters a subset of p65/p50 heterodimers, whose activation can only be initiated by IKKα [21]. IKK catalytic activity also regulates other cellular responses independent of NF-κB activation. For example, IKKβ controls anti-apoptotic, pro-inflammatory and proliferative responses by targeting p53, FoxO and other cell cycle effectors [19], [20], [22].

NF-κB is activated by moderate ROS levels in a cell-type specific manner by different mechanisms, including the classical IKKβ-dependent pathway [23], [24]. In addition, genotoxic stimuli producing DSBs cause ATM/NEMO-dependent IKKβ-mediated canonical NF-κB activation, which facilitates cell survival and cycle arrest thereby giving cells precious time to repair their damaged DNA before p53-dependent apoptotic pathways are invoked [25], [26]. A role for IKKα in DDR invoked by oxidative stress or chemotherapeutics has also been suggested [27], [28]. Collectively, these studies provided evidence that the induction of NF-κB signaling by DNA damage is important for cell survival. However, to our knowledge little if any evidence is available on how canonical NF-κB activity functionally contributes to ROS-induced HDF senescence responses.

To address NF-κB's functional roles in ROS-mediated DDR in the context of normal vs. human tumor cells, we inhibited canonical NF-κB activation in MRC-5 and IMR-90 normal HDFs and suppressed NF-κB signaling in A549 human lung tumor epithelial cells. Inhibition of canonical NF-κB in both cell types exacerbated ROS initiated DDR by up-regulating the p53–p21^Cip1 axis, leading to the enhanced accumulation of cells at the G2 or G2-M checkpoint. Importantly, the final physiological outcomes of ROS-induced DDR greatly differed in normal vs. human cancer cells with the former normal human diploid fibroblasts (HDFs) succumbing to premature senescence and the latter human lung cancer epithelial cells undergoing exacerbated apoptosis.