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• W4238740062 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression. https://doi.org/10.7554/eLife.07270.001 eLife digest Damaged DNA threatens the normal activity of living cells, so cells use a number of mechanisms to ensure that this damage is repaired. When DNA is damaged, an enzyme called ATM activates several other proteins that ultimately lead to the DNA being repaired. Problems with detecting and repairing damaged DNA have been linked to cancer. Thus, these pathways, including the activity of ATM, were previously thought to only be involved in cancer suppression. Now, Chen et al. report a new cancer-promoting role for ATM. The experiments reveal that reducing the amount of ATM in cancer cells actually made them less able to migrate and less invasive. Likewise, human breast cancer cells in which the levels of ATM had been depleted formed fewer lung tumors than normal breast cancer cells when they were transplated into mice. Oxidative stress—a build-up of harmful chemicals inside cells—is a signature feature of cancer cells and is known to be another signal that activates ATM. Chen et al. found that activating ATM through oxidative stress, but not by DNA damage, encouraged the cancer cells to migrate and become invasive. Further analysis of cellular responses following ATM activation by oxidative stress revealed that this enzyme regulates the production of a small protein called interleukin-8. This protein is an important pro-inflammatory molecule that has been implicated in cancer, in particular, in helping cancer cells to migrate to other tissues. When interleukin-8 was added to ATM-depleted cancer cells, it rescued their defects in spreading and invasiveness, thereby providing strong evidence that interleukin-8 is a biologically important target of ATM. Clinical data confirmed that breast cancer cells that had also spread to the patient's lungs often produced high levels of interleukin-8. Together, these findings suggest that ATM could be a potential target for anti-cancer therapies, as inhibiting this enzyme would inhibit interleukin-8, and in turn slow the progression and spread of cancer. https://doi.org/10.7554/eLife.07270.002 Introduction Cancer treatments rely heavily on DNA damaging agents, including radiation and chemotherapeutics, to eliminate cancer cells and decrease tumor burden (Begg et al., 2011; Lord and Ashworth, 2012; Cheung-Ong et al., 2013). These cancer therapies activate complex signaling networks, termed the DNA damage response (DDR), that detect and signal the presence of DNA damage to promote cell cycle arrest and DNA repair (Jackson and Bartek, 2009; Ciccia and Elledge, 2010). The apical protein kinase ataxia-telangiectasia mutated (ATM) initiates a large signaling cascade in response to DNA double-strand breaks (DSBs) by phosphorylating many key proteins, including the tumor suppressor p53, to orchestrate the DDR (Shiloh and Ziv, 2013; Stracker et al., 2013). The DDR regulates cell fate decision pathways including apoptosis, senescence and differentiation. Many of these pathways depend on ATM and/or p53 to enforce tumor suppressive anti-cancer barriers to limit proliferation in response to, and in the presence of, DNA damage (Norbury and Zhivotovsky, 2004; Bartkova et al., 2005; Gorgoulis et al., 2005; Bartkova et al., 2006; Di Micco et al., 2006; Vousden and Lane, 2007; Sherman et al., 2011; Roos and Kaina, 2013). However, cancer cells exhibit genome instability (Hanahan and Weinberg, 2011) and often contain endogenous oxidative and replicative damage that can promote genetic alterations to drive malignant transformation (Klaunig et al., 2010; Hills and Diffley, 2014). Cancer cells evolve a defective DDR to allow cell proliferation in the presence of DNA damage, furthering genome instability and cancer progression. Defects in the DDR can also profoundly influence DNA damage-dependent therapies both positively and negatively (Bouwman and Jonkers, 2012). Thus, DDRs can influence both cancer promoting and suppressing mechanisms. This concept is perhaps best exemplified by p53, which is normally activated by stress signals and promotes tumor suppressor pathways (Bieging et al., 2014). However, the tumor-associated gain-of-function p53 mutations that are most commonly observed in human cancers exhibit several tumor-promoting functions (Muller and Vousden, 2013). Thus, tumor suppressive or promoting activities from the same protein can be dictated by many factors including mutations, cell type, and/or disease context. ATM regulates complex signaling networks that are involved in many biological processes in addition to DSB signaling and repair (Stracker et al., 2013). Aside from increased tumorigenesis, ATM-deficiency results in altered metabolism, aberrant immune and inflammatory responses and increased levels of reactive oxygen species (ROS, Schneider et al., 2006; Alexander et al., 2010; Freund et al., 2011; Kulinski et al., 2012; Valentin-Vega et al., 2012). Several of the pathological outcomes in ATM deficient mice have been linked to ROS and many of these pathologies can be reversed by the addition of antioxidants, highlighting the important role of ATM in regulating redox-homeostasis (Ito et al., 2004; Reliene and Schiestl, 2006, 2007; Reliene et al., 2008; Freund et al., 2011; Okuno et al., 2012; D'Souza et al., 2013). In addition, ATM can be directly activated by ROS, independently from DSB signaling, and has been implicated in mitochondrial quality control, potentially through an ability to localize to mitochondria (Guo et al., 2010; Valentin-Vega et al., 2012). The substrates of ATM following ROS mediated activation, and how distinct they are from those modified following DNA damage, remains unknown. The NF-κB family of transcription factors are similarly activated by multiple stimuli that include DNA damage and ROS. NF-κB signaling regulates inflammation and is involved in cancer where it has been associated, like p53, with both anti and pro-tumorigenic processes (Ben-Neriah and Karin, 2011; Oeckinghaus et al., 2011). NF-κB signaling can be regulated by ATM through the phosphorylation of NEMO, which aids in transmitting nuclear ATM signaling to activate NF-κB in the cytoplasm (Wu et al., 2006). Upon activation, NF-κB translocates to the nucleus where it regulates gene expression and influences cell survival pathways. Understanding the crosstalk between the NF-κB and ATM signaling in complex pathologies including inflammation and cancer remains an area of active investigation. Cancer cells acquire several capabilities in addition to uncontrolled proliferation during the multistep process of tumorigenesis, including cell migration and invasion that promote tumor metastasis. (Friedl and Alexander, 2011; Hanahan and Weinberg, 2011; Valastyan and Weinberg, 2011). Metastasis occurs when a tumor spreads by means of cell migration and invasion to a secondary site, a process linked with the majority of cancer deaths (Hanahan and Weinberg, 2011). Several pathways suppress this dangerous process as part of tumor suppressive mechanisms (Mehlen and Puisieux, 2006). Although the role of DNA damage and the DDR in this process has not been extensively explored experimentally, increased genomic instability due to the acquisition of DDR defects has been proposed to play a role in the acquisition of these traits based on human patient data (Halazonetis et al., 2008). Here, we studied the contribution of the DDR kinase, ATM, in metastatic programs operating in cancer cells. We found that ATM supports migration and invasion, cellular processes intimately linked with metastasis. Our data is consistent with endogenous oxidative stress triggering these responses independently from DSB signaling. Gene expression analysis identified an ATM and mutant p53 transcriptional program that contained pro-migration and invasion genes, including interleukin-8 (IL-8). Addition of IL-8 rescued defects in cell migration and invasion observed in ATM-depleted cells thus validating this target. Consistent with the biological relevance of these findings, ATM promoted tumor formation in a xenograft human breast cancer cell model. Based on the data, we propose a non-canonical role for ATM in supporting pro-tumorigenic behavior of cancer cells. Results ATM promotes cell migration and invasion independent of DNA DSBs Given the prevalence and success of DNA-damage based cancer treatments, we sought to determine whether DNA damage and the DDR could inhibit pro-metastatic processes as part of its well-established role as an anti-cancer barrier. To this end, we first determined the effects of DNA damaging agents on cell migration, a process intimately linked with metastasis. DNA damage, including ionizing radiation (IR), topoisomerase II poisons etoposide (Et) and doxorubicin (Dox), had little effect on cell migration in the highly metastatic human breast cancer cell line MDA-MB-231 as measured by wound healing assays (Figure 1A, quantified in Figure 1B). Induction of the DNA damage marker, phosphorylated histone variant H2AX (γH2AX), confirmed DNA DSBs formations (Figure 1B). These treatments arrested the cell cycle and inhibited proliferation (Figure 1C,D). Thus, cell migration is independent from DSB induction by DNA damaging agents, as well as cell cycle and growth arrest. Figure 1 with 1 supplement see all Download asset Open asset Ataxia-telangiectasia mutated (ATM) is required for cell migration and invasion in MDA-MB-231 cells. (A) Wound-healing assays of MDA-MB-231 cells untreated (Unt) or treated with doxorubicin (Dox, 100 nM), phleomycin (Phleo, 90 μg/ml), etoposide (Et, 40 μM) or ionizing radiation (IR, 20 Gy). Drug treatments were for 48 hr. IR treatment was performed at time 0. All samples were analyzed post-48 hr from wound induction. Images were acquired at 0 and 48 hr. Representative images from three independent experiments are shown. (B) Verification of DNA damage induction and quantification of wound healing from (A). Top: Western blot analysis of samples from (A) with the DNA damage marker γH2AX. H2AX is a loading control. Bottom: Quantification of wound healing experiments from (A). (C) Cell cycle analysis of cells treated in panel A by flow cytometry. Cells were treated as in (A) and analyzed by FACS 48 hr post-treatment. (D) Proliferation of cells treated as in (A). After 48 hr, cells were trypsinized, counted and normalized to untreated cells at 0 hr. (E) ATM promotes cell migration in the absence of induced double-strand breaks (DSBs). Wound-healing assays were performed in siRNA-treated MDA-MB-231 human breast cancercells with siNon-coding (siNC) or siATM siRNAs. (F) Verification of DNA damage induction and quantification of wound healing from (E). Top: Western blot analysis of samples from (E) with the ATM markers KAP1-pS824 and mut-p53-pS15. Unmodified proteins are loading controls and ATM controls siRNA depletion. Bottom: Quantification of wound healing experiments from (E). (G) ATM depletion impairs cell migration and invasion, but not proliferation. Left panel: siNC or siATM cells were analyzed with xCELLigence Real-time cell analyzer (RTCA) to measure proliferation, migration and invasion in parallel and real-time. Experiments performed as detailed in ‘Materials and methods’. Right panel: ATM depletion 96 hr post-transfection. (mean ± s.e.m., n = 3). https://doi.org/10.7554/eLife.07270.003 Defects in DDR genes and DNA damage signaling affect cellular responses to chemotherapeutic drugs in some cancer cells (Bouwman and Jonkers, 2012). As ATM mediates many initial DNA damage signaling events and regulates multiple cellular responses to DNA damage, we tested its role in cell migration. Strikingly, ATM depletion reduced cell migration in the presence or absence of IR-induced damage (Figure 1E, quantified in Figure 1F). Increased DDR signaling as evidenced by phosphorylated H2AX, p53 and KAP1 confirmed DNA damage by IR (Figure 1F). We independently verified the requirement of ATM for cell migration and invasion using an xCelligence system that measures cell migration and invasion, as well as proliferation in real-time and in the same cell populations which provides standardized experimental conditions. Reduced invasion and migration in ATM-depleted cells was independent from proliferation as control and ATM-depleted cells grew similarly (Figure 1G). Experiments with two additional independent ATM siRNAs confirmed these observations thus ruling out possible off-target effects by siRNAs (Figure 1—figure supplement 1). Thus, ATM promotes cell migration and invasion in MDA-MB-231 cells in the absence of exogenous DNA damage. ATM and p53 operate to promote cell migration and invasion ATM is activated by at least two distinct mechanisms (Shiloh and Ziv, 2013). DSBs activate ATM through interactions with the MRE11-RAD50-NBS1 (MRN) complex. ATM phosphorylates many targets, including the effector kinase CHK2, which collectively orchestrate the DDR (Bakkenist and Kastan, 2003; Lee and Paull, 2005; Matsuoka et al., 2007; Shiloh and Ziv, 2013). ATM is also activated by oxidative stress independently of DSBs, MRN or CHK2 (Guo et al., 2010), although the downstream pathways reliant on this ATM activation are unclear. In contrast to ATM inhibition, depletion of CHK2, MRE11 or NBS1 did not reduce cell migration, suggesting ATM-mediated cell migration occurred independently of DSB-dependent ATM activation (Figure 2A,B; depletion analysis shown in Figure 1C). Thus, we hypothesized that ATM could promote migration through oxidative stress. In support of this idea, chemical inhibition of oxidative stress in MDA-MB-231 cells using the reducing agent N-acetylcysteine (NAC) reduced cell migration similarly as depletion of ATM (Figure 2D and Figure 2—figure supplement 1). We also showed that NAC treatment inhibits intracellular oxidative stress (Figure 2E). To rule out any influence of proliferation on cell migration in our analyses, we performed live-cell imaging to track the migration of individual cells. We found reduced cell motility in cells treated with NAC compared with control cells (Figure 2F,G and Videos 1, 2). These data suggest that ATM, independent from DSB signaling, promotes cell migration. Figure 2 with 1 supplement see all Download asset Open asset ATM promotes cell migration and invasion independently of DNA DSB signaling in MDA-MB-231 cells. (A) DSB signaling is not involved in cell migration. Experiments were performed as in Figure 1A with the indicated siRNAs. (B and C) Quantification of wound healing (B) and siRNA depletions (C) in (A). (D) Reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) reduces cell migration. Right panel: quantification of wound healing. (E) NAC treatment reduces endogenous ROS. Cells treated with 10 mM NAC were analyzed using an intracellular ROS detector as detailed in ‘Materials and methods’. 4 mM H2O2 treatment serves as a positive control. (F) Live-imaging analysis of cells treated with 10 mM NAC or left untreated. Images were acquired every 15 min for 6 hr and cell were tracked using ImageJ. Colored dots and lines represent individual cell paths. Scale bar, 37.5 μm. (G) Quantification of individual cell speed (μm/min) and cell path (μm) from (F). Cell parameters were quantified in ImageJ and represent mean data from >100 cells. Error bars = SD. *** p-value <0.0001, unpaired two-tailed t-test. https://doi.org/10.7554/eLife.07270.005 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live cell imaging and tracking of untreated MDA-MB-231 cells for Figure 2. Images were taken every 15 min for 6 hr and tracking was performed in ImageJ. Still images and quantifications are provided in Figure 2E,F. https://doi.org/10.7554/eLife.07270.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live cell imaging and tracking of MDA-MB-231 cells treated with 10 mM NAC for Figure 2. Images were taken every 15 min for 6 hr and tracking was performed in ImageJ. Still images and quantifications are provided in Figure 2E,F. https://doi.org/10.7554/eLife.07270.008 While DSBs trigger ATM-dependent phosphorylation of more than 1000 proteins (Matsuoka et al., 2007; Bennetzen et al., 2010; Bensimon et al., 2010), very few targets of ATM in response to other types of damage, including oxidative stress, are known (Guo et al., 2010). However, p53 is phosphorylated by ATM after oxidative stress and mutant p53 is involved in TGFβ-dependent cell migration in MDA-MB-231 cells (Adorno et al., 2009; Guo et al., 2010). We therefore speculated that mutant p53 could act in concert with ATM to promote cell migration. To test this hypothesis, we depleted ATM, mutant p53 or both and analyzed cell migration. As expected, inhibition of mutant p53 resulted in reduced cell migration (Figure 3A, quantified in Figure 3B). Interestingly, co-depletion of ATM and mutant p53 resulted in a similar reduction of cell migration as either single gene knockdown alone (Figure 3A, quantified in Figure 3B). We confirmed and extended these results using real-time, simultaneous analyses of proliferation, migration and invasion. Although mutant p53 depletion mildly reduced proliferation, co-depletion of ATM resulted in an epistatic reduction of cell migration and invasion (Figure 3C). Live-cell imaging of migrating cells revealed reduced speed and migration path length in ATM and mutant p53 depleted cells (Figure 3D,E and Videos 3–5). These results further corroborate the role of ATM and mutant p53 in promoting cell migration, independently from cell proliferation. These results are consistent with our analyses of DNA damaging agents, which inhibit proliferation without altering cell migration (Figure 1A–D). We repeated these experiments in the highly metastatic BT-549 breast cancer cells that have mutant p53. Consistent with results from MDA-MB-231 cells, depletion of ATM reduced cell migration by ∼ 60% in BT-549 (Figure 3—figure supplement 1A). ATM and/or mutant p53-depleted BT-549 cells exhibited similar levels of migration (Figure 3—figure supplement 1B), which is in accord with data obtained in MDA-MB-231 cells (Figure 3A–C). Taken together, these results from multiple human cancer cell lines suggest that ATM and mutant p53 are required for the cell migration and invasion phenotypes observed in these highly invasive cancer cell lines. Figure 3 with 1 supplement see all Download asset Open asset ATM-mutant p53 axis of the DNA damage response (DDR) promotes cell migration and invasion in MDA-MB-231 cells. (A) ATM or mutant p53 depletion, as well as co-depletion, impairs cell motility similarly. Wound-healing assays were performed with the indicated siRNAs as in Figure 2A. (B) siRNA depletions and quantification of wound healing for (A). (C) Real-time analysis of cell dynamics in siATM, simutant-p53 and co-depleted cells. Experiments performed as in Figure 1G with indicated siRNAs. Right: ATM and mutant p53 levels in cell samples. (D) Live cell imaging of cell migration defects in ATM and mutant p53 depleted MDA-MB-231 cells. Experiments were performed and analyzed as in Figure 2F. Scale bar, 20 μm. (E) Quantification of individual cell speed (μm/min) and cell path (μm) from (D). Cell parameters were quantified as in Figure 2G. Error bars = SD. *** p-value <0.0001, unpaired two-tailed t-test. https://doi.org/10.7554/eLife.07270.009 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live cell imaging and tracking of siNC MDA-MB-231 cells for Figure 2. Images were taken every 15 min for 6 hr and tracking was performed in ImageJ. Still images and quantifications are provided in Figure 3D,E. https://doi.org/10.7554/eLife.07270.011 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live cell imaging and tracking of siATM MDA-MB-231 cells for Figure 3. Images were taken every 15 min for 6 hr and tracking was performed in ImageJ. Still images and quantifications are provided in Figure 3D,E. https://doi.org/10.7554/eLife.07270.012 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live cell imaging and tracking of si mutant p53 MDA-MB-231 cells for Figure 3. Images were taken every 15 min for 6 hr and tracking was performed in ImageJ. Still images and quantifications are provided in Figure 3D,E. https://doi.org/10.7554/eLife.07270.013 ATM regulates interleukin-8 (IL-8) We next investigated the molecular mechanism by which ATM promotes cell migration and invasion. The identification of an epistatic relationship between ATM and mutant p53 in promoting cell migration pointed towards a common molecular pathway. Given the role of mutant p53 in transcriptional regulation, we hypothesized that ATM could similarly regulate the expression of genes involved in cell migration (Riley et al., 2008; Shiloh and Ziv, 2013). Therefore, we performed a microarray-based comparative gene expression analysis in control, ATM and mutant p53 siRNA-treated cells. Out of the hundreds of genes in ATM-depleted cells whose mRNA levels were differentially regulated more than 1.5-fold compared to control siNon-coding (siNC) cells, only ∼40 genes showed equivalent regulation between siATM and si mutant p53 cells (Figure 4A and Supplementary file 1A,B). We observed comparable numbers of co-regulated genes that were either up-regulated or down-regulated similarly in both siATM and si mutant p53 cells (Figure 4A and Supplementary file 1C). Gene ontology (GO) analysis indicated that genes involved in the response to wound healing, including cell migration genes, were in the top 10 GO categories for both ATM and mutant p53 depleted samples (Figure 4—figure supplement 1). GO analysis of genes co-regulated by ATM and mutant p53 identified almost exclusively pathways involving cell migration (Figure 4—figure supplement 1C and Supplementary file 1C) in agreement with our data showing reduced migration in ATM or mutant p53 deficient cells. Collectively these data support a role for mutant p53 and ATM in the co-regulation of a gene network regulating cell migration under these conditions. Figure 4 with 2 supplements see all Download asset Open asset ATM-mutant p53 regulates cytokine interleukin-8. (A) Differential transcriptome expression analysis in siATM- and simut-p53- depleted cells identifies reduced IL-8 expression in both samples. Upper: Venn diagram of differentially expressed genes in simut-p53, siATM or both. Numbers indicate genes differentially expressed 1.5-fold or greater compared to siNC. Heatmap represents the 38 genes co-regulated similarly in siATM and simut-p53 cells. Expression data was normalized to control siNC cells. Cut-off = 1.5-fold normalized to siNC control cells. (B and C) qRT-PCR analysis of IL-8 mRNA levels in MDA-MB-231 (B) and BT-549 (C) siATM or sipmut-53 depleted cells. (D) IL-8 promoter activity by luciferase reporter assay in siATM and simut-p53 cells. Depletion of (E) NF-κB or (F) NEMO impairs IL-8 expression. (G) ATM or mutant p53 depletion abrogates NF-κB p65 nuclear localization. Cells treated with indicated siRNA were harvested to obtain cytoplasmic extract (CE) and nuclear extract (NE) to analyze NF-κB p65 localization. (H) ATM or mutant p53 deletion impairs NF-κB p65 binding to IL-8 promoter using chromatin immunoprecipitation (ChIP) analysis. Actin promoter serves as a negative control. https://doi.org/10.7554/eLife.07270.014 We focused our analysis on the down-regulated genes identified in both ATM and mutant p53 data sets since GO analysis only identified the wound healing pathway in this gene set (Figure 4—figure supplement 1). Interestingly, the inflammatory cytokine interleukin-8 (IL-8) was the most down-regulated gene in both siATM and si mutant p53 cells (Figure 4A). We confirmed IL-8 down-regulation in mutant p53-containing cell lines MDA-MB-231 and BT-549 upon ATM depletion (Figure 4B,C). Conversely, depletion of ATM in cancer cell lines containing WT p53 resulted in increased IL-8 mRNA levels (Figure 4—figure supplement 2A,B). These results suggest that ATM promotes IL-8 levels in the context of mutant p53. IL-8 is upregulated in several cancers, including breast cancer, where it mediates several cancer promoting pathways including cell migration (Campbell et al., 2013; Singh et al., 2013). The IL-8 promoter contains many transcription factor binding sites, including NF-κB, which regulates IL-8 expression and is linked to the DDR through ATM activation by DSBs (Mukaida et al., 1990; Biton and Ashkenazi, 2011; McCool and Miyamoto, 2012). We confirmed IL-8 promoter regulation by NF-κB as ∼90% of IL-8 promoter activity was lost by mutating the NF-κB binding site (mut IL-8, Figure 4D). Interestingly, depletion of ATM or mutant p53 reduced IL-8 promoter activity similarly as mut IL-8, showing ATM regulation of IL-8 occurs at the transcriptional level (Figure 4D). As expected, we observed that depletion of NF-κB p65, a subunit of NF-κB dimer, or NEMO abrogated IL-8 expression in MDA-MB-231 (Figure 4E, Freund et al., 2004). Both ATM and p53 are known to be required for NF-κB localization and activation in the nucleus upon various stimuli including cellular stress (Wuerzberger-Davis et al., 2007; Hoesel and Schmid, 2013). To determine whether NF-κB function required ATM or mutant p53 in our cell system, we investigated the nuclear localization of the NF-κB subunit p65 in MDA-MB-231 cells under normal growth conditions. Nuclear localization of the p50/p65 NF-κB dimer enables transcriptional activation of this complex so we analyzed p65 nuclear accumulation as a readout of NF-κB localization (Hayden and Ghosh, 2012). We observed reduced p65 nuclear localization and NEMO phosphorylation in ATM- and mutant p53-depleted cells compared to control cells, which is inline with the reduced IL-8 expression that occurs under these conditions (Figure 4G, Figure 4—figure supplement 2F). We next performed chromatin immunoprecipitation (ChIP) of NF-κB on the IL-8 promoter to analyze directly the involvement of NF-κB in regulating IL-8 transcription and how this is affected by ATM and mutant p53. ChIP analyses revealed that reduced levels of ATM or mutant p53 impaired NF-κB accumulation on the IL-8 promoter (Figure 4H). Collectively, our results strongly suggest that ATM and mutant p53 are required for NF-κB activity, which is necessary to regulate IL-8 expression. Further analyses supported the notion of IL-8 as the gene responsible for reduced migration in ATM-depleted MDA-MB-231 cells as (1) IL-8 depletion reduced cell migration and invasion, (2) NAC treatment reduced IL-8 mRNA levels and (3) oxidative stress induction by H2O2 increased IL-8 levels and (4) H2O2-induced IL-8 expression was dependent on ATM (Figure 5A–E). Taken together, these results suggest that ATM regulates a transcriptional network that includes the NF-κB-regulated gene IL-8. Our data suggests that this ATM pathway promotes cell migration and invasion in MDA-MB-231 cells through a cell intrinsic mechanism that is reliant on endogenous oxidative stress. Figure 5 with 1 supplement see all Download asset Open asset ATM promotes pro-metastatic IL-8-dependent cellular processes. (A) IL-8 depletion reduces cell migration and invasion. Experiments performed as in Figure 1G. Error bars = SEM. * p-value <0" @default.
• W4238740062 created "2022-05-12" @default.
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• W4238740062 title "Decision letter: ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion" @default.
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