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Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress Caitlin E. Tolbert a, Matthew V. Beck b, Claire E. Kilmer c, Melissa C. Srougi b, * a
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA Department of Chemistry, High Point University, High Point, NC, 27268, USA c Biotechnology Program, North Carolina State University, Raleigh, NC, 27607, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2018 Accepted 5 December 2018 Available online xxx
Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is integral in the response to DNA double-stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM-deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor-driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N-Acetyl-L-cysteine restores wild-type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro-migratory cellular behavior. © 2018 Elsevier Inc. All rights reserved.
Keywords: ATM Rac1 Reactive oxygen species Migration
1. Introduction Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by immunodeficiency, progressive cerebral ataxia, oculocutaneous telangiectasia, and a high incidence of cancer [1]. Individuals with A-T have ~30e40% cumulative incidence of cancer by age 40, primarily lymphoma and leukemia although breast, gastric, and other solid tumors also occur at high rates [2]. A-T is caused by a mutation in the A-T-mutated (ATM) gene. ATM is one of the most commonly mutated genes in lung adenocarcinoma, pancreatic cancers, and hematologic malignancies, while methylation or deletion of ATM has been reported in breast cancers. Functionally, ATM is a serine/threonine kinase related to the PI3Klike family of kinases. ATM is integral to the DNA damage response (DDR) pathway and is activated by double-stranded breaks (DSBs) such as after ionizing radiation (IR) exposure [3]. Upon activation by DSBs, ATM promotes DNA repair via cell cycle checkpoint activation.
* Corresponding author. Department of Chemistry, High Point University, One University Parkway, Couch Hall Rm 355D, High Point, NC, 27268, USA. E-mail address: [email protected] (M.C. Srougi).
Interestingly, A-T patients and ATM-/- cells exhibit high levels of oxidative stress, which may contribute to insulin resistance, neurodegeneration, and cancer progression observed in A-T [4,5]. A growing body of research suggest that mitochondrial dysfunction may contribute directly to elevated oxidative stress in A-T cells. These studies highlight a reduction in total mitochondrial respiratory activity, membrane potential (D4m), and expression of ROS detoxifying genes resulting in mitochondrial ROS overproduction and decreased ATP levels [6,7]. Given this context, ATM has a noncanonical role as a redox-sensor that maintains cellular redox homeostasis by modulation of antioxidant levels [8]. In ATM-null mice, several cancer types and neuromotor functions have improved with antioxidant treatment, which restore cellular redox homeostasis and D4m [9e11]. However, a direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Due to its role in regulating actin reorganization and cell migration, Rac1 GTPase is a critical component of tumorigenesis and invasiveness. Rac1 and other Rho GTPase family members are overexpressed in some human cancers however, unlike the related Ras GTPases, they are not activated by oncogenic mutations [12]. Instead, increased Rac1 activity may occur by altering the activity of
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Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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regulatory proteins. As with all canonical GTPases, activity is based on its nucleotide bound state: GTP-bound protein is “on” versus GDP-bound protein is “off”. Guanine nucleotide exchange factors (GEFs) facilitate the GTP-bound activated state, which signal to downstream effectors to alter cellular behavior. Besides regulatory proteins, some GTPases can be directly activated by ROS and reactive nitrogen species (RNS) in the absence of GEFs. Studies have identified a redox-sensitive motif in the phosphoryl-binding loop of Rho family members. Oxidation of reactive cysteines (Cys16/Cys20) within this motif destabilizes the bound nucleotide, allowing binding of GTP due to its excess in the cytosol. Treatment of recombinant GTPase with superoxide anion radical or nitrogen dioxide radicals in vitro induced guanine nucleotide dissociation, which could be reversed by radical quenching agents [13,14]. Further studies demonstrated direct activation of RhoA by ROS, causing actin cytoskeleton rearrangements [15]. Similar to RhoA, in vitro studies with Rac1 have shown that both ROS and RNS alter the charge of Cys18 leading to nucleotide exchange and subsequent activation of Rac1 [13,16]. Here, we report that cells lacking ATM kinase activity exhibit altered cytoskeletal dynamics, enhanced cell migration, and increased Rac1 activity. Loss of Rac1 in ATM-null cells decreased cell migration to levels similar to cells expressing wild-type ATM. Further investigation revealed that activation of Rac1 in ATM-null cells was due to enhanced ROS generation. Antioxidant treatment not only abrogated Rac1 activity but also reverted cell migration back to wild-type levels. These studies identify and highlight a role for Rac1 in promoting migration under conditions of elevated oxidative stress such as with ATM loss, which may facilitate tumorigenesis and metastasis noted in A-T patients and ATMmutated cancers. 2. Materials and methods 2.1. Reagents and antibodies CM-H2DCFDA was obtained from Molecular Probes (Eugene, OR). The Rac1 antibody (Clone 102), anti-Paxillin (Clone 165) were from BD Biosciences. Anti-GAPDH antibody was from SigmaAldrich (St. Louis, MO). Alexa Fluor 594 phalloidin and goat antimouse Alexa Fluor 488 were obtained from Molecular Probes. Peroxidase-conjugated goat anti-mouse and goat anti-rabbit were from Santa Cruz Biotechnology. Fibronectin (FN) was from Fisher Scientific.
camera and acquired using Metamorph Workstation (Universal Imaging Corp., META-40002). Cell morphology analysis was performed using NIH Image J software. Stress fiber (SF) formation was quantified by manual counting of cells containing at least one SF that transversed the cell. Cell circularity was defined as cell area divided by cell perimeter. Values ranged from 0 to 1.0 indicating an elongated or rounded morphology, respectfully. Actin-enriched ruffles were quanitifed from phalloidin stained cells. 2.4. Rac1 activity assays and western blotting Plated cells were washed with ice-cold PBS, lysed on ice, and assayed for Rac activation by glutathione S-transferase-p21activated kinase pulldown, as described by Sander et al. [17]. Bead fraction (active Rac) and total cell lysates (total Rac) were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and immunoblotted with the indicated primary and HRP-secondary antibodies. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and visualized using a BioRad ChemiDoc system. For quantification of western blots, intensity of bands from three separate experiments was obtained using Image J. 2.5. RTCA and cell spreading on fibronectin Assays were performed as previously described [18]. Briefly, cells were serum-starved for 3e16 h in high glucose DMEM/0.5% delipidated BSA (Sigma-Aldrich) prior to trypsinization and being held in suspension for 2 h in the same media. The xCELLigence RealTime Cell Analyzer (RTCA) system (Acea Biosciences/Roche Applied Science 05-469-759-001) was used to measure electrical impedance. Wells were coated with 50 mg/mL FN, then blocked with DMEM/0.5% delipidated BSA before seeding 2000 cells per well. The RTCA system uses electrical impedance signals, expressed as “cell index” (CI), to monitor cell adhesion and spreading of cells grown directly on microelectrode-coated surfaces. CI was recorded every 15 min for 4 h. Experiments were repeated three independent times. 2.6. ROS generation Formation of ROS was monitored by the conversion of nonfluorescent 5-(and-6)-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) to fluorescent DCF according to manufacturer's instructions (Invitrogen).
2.2. Cell culture GM16667 (ATMþ/þ cells) and GM16666 (ATM-/- cells) were from the Coriell Institute (Camden, NJ). HeLa and REF52 cells were from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in high glucose-containing DMEM with 10% fetal bovine serum (FBS), or 15% FBS (for ATM cells), 100 mg/mL hygromycin (for ATM cells), 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 C in a 5% CO2 humidified atmosphere. All tissue culture components were from Invitrogen (Carlsbad, CA). KU55933 (KU) and N-Acetyl-L-cysteine (NAC) were from Sigma. 2.3. Immunofluorescence Coverslips were fixed in 3.7% paraformaldehyde and permeabilized in 0.2% Triton X-100/phosphate-buffered saline (PBS) prior to staining with indicated primary and secondary antibodies. Immunofluorescence images were taken with a Zeiss axiovert 200M microscope equipped with a Hamamatsu ORCA-ERAG digital
2.7. siRNA transfection HeLa cells at ~60% confluence were transfected with nontargeting siRNA (siControl) or siRNA targeted to Rac1 (siRac1) (Dharmacon) at 25 nM using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Wound healing assays were performed 48 h after transfection. 2.8. Wound healing assays Cells were plated on 30 mg/mL FN-coated coverslips and grown to confluency. Three parallel scratches were made on the cell monolayer using a 200 mL pipette tip, and media was replaced with 2% FBS-containing media. Where indicated, medium was supplemented with 10 mM KU55933, 0.5 mM NAC or DMSO. Live-cell images were taken of 4 random fields along the scratch at 0 and 5 h after wounding using an Olympus IX73 microscope (Olympus, Center Valley, PA) equipped with a CMOS 16 bit camera. Wound width was quantified using NIH Image J and represented as average
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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% wound closure in 5 h from the initial width at 0 h. 2.9. Single-cell random migration For live single-cell tracking, cells were sparsely seeded onto 10 mg/ml FN-coated glass bottom culture dishes (MatTek Corporation) for 4e6 h prior to imaging. Time-lapse microscopy was performed with a Nikon BioStation IM (20x objective). Images were acquired every 10 min for 12 h. Cell velocity, persistence, and distance migrated was measured with NIH ImageJ using the Manual Tracking plug-in (http://rsbweb.nih.gov/ij/plugins/track/track. html). 3. Results 3.1. Loss of ATM kinase activity increases cellular spreading and Rac1 activation To determine if ATM activity alters cytoskeletal dynamics we first examined the spreading ability of ATM-inhibited cells. When cells are held in suspension for 2 h and then re-plated onto FN, coordinated control of actin cytoskeletal remodeling by several members of the Rho family of GTPases occurs. Cells change morphology from round to flattened with numerous matrix adhesions. These changes are attributed to the biphasic activation of RhoA and Rac1, with Rac1 promoting cell ruffling and spreading [19]. Due to tight control during cell spreading, this process provides a readout for perturbations in GTPase activity. REF52 cells were serum-starved and then held in suspension for 2 h while treated with the selective ATM kinase inhibitor KU55933 (KU). Once plated on FN, KU-treated cells displayed altered cytoskeletal rearrangement kinetics, displaying enhanced stress fibers (SFs) by 15 min compared to vehicle -treated cells (Fig. 1A). A significant decrease in cell circularity was noted in KU-treated cells suggesting increased radial spreading as cells begin to adhere (Fig. 1B). Increased SF formation and actin-enriched ruffles were also observed in KU-treated cells compared to vehicle-treated cells. (Fig. 1A and 1C-D). These observed changes in cytoskeletal dynamics during early spreading events suggested altered Rho GTPase activity. Therefore, we examined basal Rac1 activation in HeLa cells treated with KU. We observed that serum-starved HeLa cells pretreated with KU exhibited a significant 2-fold increase in active Rac1 compared to vehicle-treated cells (Fig. 1E). A significant 2-fold increase in active Rac1 was also confirmed in ATM-/- human fibroblasts derived from patients with A-T compared to an isogenic cell line re-expressing wild-type ATM (ATMþ/þ) (Fig. 1F). Furthermore, the increase in Rac1 activity was confirmed in HeLa cells transfected with a kinase-dead ATM construct (Supplementary Fig. 1). Together, these studies suggest that loss of ATM kinase function not only alters cytoskeletal dynamics, but also increases basal Rac1 activity. 3.2. Loss of ATM kinase activity increases cellular spreading and migration To evaluate the role of ATM in the long-term dynamics of cellular spreading and migration, we used the ATM-/- and ATMþ/þ human fibroblast model. Upon plating on FN, ATM-/- fibroblasts exhibited enhanced SF formation by 20 min. In contrast, ATMþ/þ cells showed SF formation beginning at 30 min (Supplementary Fig. 2), consistent with our previous findings (Fig. 1AeD). Rac1 activation remained high in ATM-/- cells from 0 to 20 min after adhesion, and dissipated by 30 min, the time when SF formation peaked (Fig. 2A and Supplementary Fig. 2). Conversely, ATMþ/þ cells showed characteristic biphasic activation of Rac1 where levels
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started high, decreased upon adhesion, and then increased again at 30 min (Fig. 2A) [19]. Since Rac1 is significantly activated upon loss of ATM kinase activity, we wanted to monitor the downstream effects on cellular spreading and migration. To do this, we used the automated RTCA (Real-Time Cell Analysis) system to quantify cell spreading [20]. ATMþ/þ and ATM-/- fibroblasts were plated at sub-confluency on FN-coated microelectrode E-Plates and impedance was analyzed over time. As shown in Fig. 2B (left panel), cell index (CI) was increased with ATM-/- cells compared to control ATMþ/þ cells, suggesting increased cell spreading. Loss of ATM significantly increased CI at both 2 h and 4 h compared to cells expressing wildtype ATM (Fig. 2B, right panel). We next evaluated the long-term effects of altered cytoskeletal dynamics on cell motility using time-lapse microscopy of ATMþ/þ and ATM-/- cells grown on FN. ATM-/- cells migrated significantly faster (0.6 mm/min ± S.D. 0.02) than ATMþ/þ cells (0.3 mm/ min ± S.D. 0.04) (Fig. 2C). ATM-/- cells also migrated significantly farther compared to ATMþ/þ cells, with a mean distance of ~130 mm (þ/ S.D. 32) versus 80 mm (þ/ S.D. 30), respectively (Fig. 2D). Together, these data suggest that loss of ATM results in Rac1 activation, increased cell spreading, and enhanced motility compared to wild-type cells. 3.3. Enhanced cell migration in ATM-inhibited cells requires Rac1 To explore the direct role of Rac1 in ATM-mediated alterations in cell migration, we knocked-down Rac1 in HeLa cells using siRNA and performed Rac1 activity and wound healing assays. HeLa cells were transfected with a non-targeting control RNAi (siControl) or RNAi targeted to Rac1 (siRac1), treated with DMSO or KU, and Rac1 activity was measured. Low Rac1 activity was observed in siControl DMSO-treated HeLa cells (Fig. 3A). In contrast, elevated Rac1 activity was detected in siControl KU-treated cells as previously observed (Fig. 1D). In siRac1 KU-treated cells, Rac1 activity was abrogated to a similar level as DMSO-treated siControl cells (Fig. 3A). Using these experimental conditions, we examined the effects of reduced Rac1 activity on cellular migration in ATMinhibited cells. Wound healing assays were performed on confluent monolayers 48 h post-transfection and the percent wound closure determined after 5 h. Migration was significantly increased in siControl KU-treated cells compared to siControl DMSO-treated (~41% versus ~23%, respectively) (Fig. 3B). This is consistent with increased Rac1 activity observed in KU-treated cells (Figs. 1E and 3A). However, siRac1 KU-treated cells exhibited a significant decrease in migration (~12%), compared to siControl KUtreated cells (Fig. 3B). Cells transfected with siRac1 and treated with DMSO had no significant changes in migration compared to siControl DMSO-treated cells (16% versus 23%, respectively). Collectively, these data suggest that Rac1 is important in the enhanced migratory phenotype noted in cells lacking ATM kinase activity. 3.4. Elevated ROS activates Rac1 in cells deficient in ATM kinase activity Previous data have shown that ATM-/- mice or A-T patients have higher levels of oxidative stress than wild-type and that this deleterious phenotype can be mitigated by treatment with the antioxidant NAC [4,21]. Furthermore, in vitro, Rho proteins can be directly activated by redox agents such as ROS and RNS [13,14]. We therefore hypothesized that loss of ATM would result in elevated ROS levels that could contribute to the activation of Rac1. We directly monitored intracellular ROS formation using the conversion of non-fluorescent CM-H2DCFDA to DCF in ATMþ/þ and ATM-/-
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 1. Inhibition of ATM kinase activity increases cell spreading and Rac1 activation. (A) Cells were plated onto FN-coated coverslips for the indicated times, then fixed and stained with phalloidin to visualize F-actin and with anti-paxillin to visualize focal adhesions. Bar, 40 mm. (B) Cell circularity (C) percentage of cells with F-actin and (D) average number of ruffles per cell were quantified for each treatment group in triplicate. Error bars indicate S.E.M. *p 0.01; **p 0.005; ***p 0.001. (E) Rac1 activity was measured by GST-PBD pulldown assay in HeLa cells treated with DMSO or KU, or (F) in ATM proficient (ATMþ/þ) or deficient (ATM-/-) human fibroblasts. For all graphs, average fold-change values from three independent experiments are represented ± S.E.M. *p 0.05.
cells. ATM-/-cells had significantly higher basal levels of ROS compared to ATMþ/þ cells (Fig. 4A). NAC treatment significantly decreased DCF fluorescence in ATM-/- cells to wild-type levels (þ/ NAC treatment) (Fig. 4A) [21], confirming that the increase in DCF fluorescence seen in ATM-/- cells was due to increased ROS generation. To determine if the elevated active Rac1 observed in ATM-/cells was due to elevated ROS, we examined Rac1 activity ± NAC. ATMþ/þ cells had low basal levels of active Rac1 with or without NAC treatment. However, the increase in active Rac1 seen in ATM-/-cells was prevented upon the addition of NAC (Fig. 4B). These data suggest that loss of ATM leads to increased susceptibility to oxidative stress, leading to Rac1 activation. Based on these findings, we postulated that antioxidant treatment should ameliorate Rac1-induced migration in ATM-/- cells. DMSO- and NAC-treated HeLa cells exhibited a wound closure of 10% and 11%, respectively, at 5 h (Fig. 4C and D). KU-treatment significantly increased wound closure (19%) compared to DMSOtreated cells (10%) (Fig. 4C and D). However, combined treatment with KU and NAC reverted wound closure to levels similar to control (7%) (Fig. 4D). Collectively, these data suggest that loss of ATM kinase activity enhances cell migration through the ROS-
mediated activation of Rac1. 4. Discussion Hereditary mutations of genes involved in DNA repair such as ATM markedly increase susceptibility to a variety of human cancers. Somatic mutations in the ATM gene are among the most commonly found aberrations and are associated with poor patient prognoses and chemotherapy resistance [2]. ATM functions not only in the maintenance of genomic integrity, but also cellular redox status. One mechanism for ATM's role in oxidative stress involves the regeneration of NADPH by promoting the pentose phosphate pathway, thus maintaining the active reduced forms of multiple detoxification proteins including thioredoxin 1, catalase, glutathione peroxidases, and superoxide dismutase [8,22]. Our data suggest that high basal Rac1 activity in cells lacking ATM kinase activity is linked to chronic oxidative stress. We demonstrate that loss of ATM kinase activity accelerates cellular spreading and migration (Fig. 1AeD and Fig. 2BeD). In ATM-/- or ATM-inhibited cells, elevated ROS levels result in increased steady-state Rac1GTP, which promoted enhanced cell motility (Fig. 4AeD). Down-
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 2. ATM deficiency enhances the kinetics of Rac1 activation, spreading, and migration upon adhesion to FN. ATMþ/þ and ATM-/- human fibroblasts were serum-starved and then held in suspension for 2 h. (A) Rac1 activity was determined upon adhesion to FN-coated dishes at indicated time points. Quantification of blots indicates the fold-change values from three independent experiments ± S.E.M. *p 0.05. (B) ATM loss enhances cellular spreading as determined by Real-time Cell Impedance Assay (RTCA). (Left panel) Representative trace of ATMþ/þ and ATM-/- human fibroblasts, graphed as Cell Index at 10 min intervals; lower Cell Index indicate decreased cellular spreading. Each data point is the average Cell Index of at least quadruplicate wells ± S.D. for each cell line. (Right panel) Graph showing relative Cell Index (cellular spreading) of ATMþ/þ and ATM-/- human fibroblasts at 2 h and 4 h. Data is the average ± S.D. combined from three independent experiments *p 0.01. (CeD) ATM deficiency increases cellular velocity and distance migrated. Singlecell live imaging was performed on ATMþ/þ and ATM-/- cells after adhesion to FN for 24 h to track (C) velocity and (D) distance traveled. Graphs represent cell averages per experiment (n ¼ 30) þ/ S.D., *p 0.05.
Fig. 3. Knock-down of Rac1 suppresses enhanced cell migration in ATM-inhibited cells. HeLa cells were transfected with a non-targeting siRNA oligo (siControl) or siRNA oligos targeted to Rac1 (siRac1). Cells were then treated with vehicle (DMSO) or KU. (A) Rac1 activity was assessed by GST-PBD pulldown assay. (B) Wound healing assay. Confluent cell monolayers were scratched with a micropipette tip. The average percent wound closure after 5 h was calculated from three independent experiments. *p 0.01, **p 0.001, not significant (NS).
regulation of active Rac1 using siRNA or the free radical scavenger NAC, reverted cells back to a reduced migration phenotype similar to wild-type cells (Figs. 3B and 4D). These data demonstrate for the first time a physiological linkage between ATM status and the regulation of Rac1 activity, which could have important therapeutic implications especially in human cancers.
Work by Heo et al. [13,14] demonstrated that ROS and RNS can directly activate Rho GTPase family members, in the absence of GEFs. In this context, activation of the GTPase can be reverted by a reducing environment or the presence of a redox scavenger (Fig. 4B). Based on our findings, we speculate that in cases of increased physiological oxidative stress, such as occurs with ATM
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 4. Increased ROS in ATM-deficient cells activates Rac1 and increases cell migration. Confluent monolayers of ATMþ/þ and ATM-/- human fibroblasts were loaded with CMH2DCFDA and then treated ± the free radical scavenger N-Acetyl-L-cysteine (NAC). Generation of ROS was monitored by the fluorescence intensity of DCF. Graph represents average from quadruplicate wells of n ¼ 3 independent experiments ± S.E.M; *p 0.05. NAC blocks Rac1 activation and enhanced migration in cells lacking ATM kinase activity. (B) (Left panel) ATMþ/þ and ATM-/- cells were treated ± NAC and Rac1 activity was assessed using GST-PBD pulldown assay. (Right panel) Fold-change in active Rac1, where the graph represents the average from three independent experiments ± S.E.M.; *p 0.05. (C) Phase contrast images of HeLa cell monolayers treated with DMSO, KU, NAC, or both NAC and KU at 0 and 5 h after wounding. (D) Quantification of the average percent wound closure after 5 h was calculated from three independent experiments; *p 0.01, **p 0.001.
loss, ROS-mediated regulation of Rho GTPases may dominate. However, we do not discount that parallel GEF-independent and -dependent pathways may be involved in an ATM-deficient/ mutated background. Therefore, ongoing research will investigate the contribution of GEF-mediated activation of Rac1 under physiological oxidative stress. Clinically, ROS may increase tumor metastasis by promoting epithelial-mesenchymal transition, migration and invasion of tumor cells, and angiogenesis [23]. Rho family GTPases are also implicated in the invasiveness of various cancers. Elevated Rac1 activity in this context would support cancer metastasis and vascularization by promoting directed migration (Fig. 4C and D). Not surprisingly, carriers of biallelic mutations responsible for A-T have a two-fold higher risk of developing cancer, especially breast cancer, compared to monoallelic mutation carriers. Furthermore, down-regulation of ATM expression in somatic breast carcinomas is correlated with increased microvascularization [11]. Our data uncover an important pathophysiological role for ROS-induced Rac1 activity in the absence of functional ATM. Previous findings have shown the benefits of NAC treatment in ATM-deficient model systems to counteract the effects of ROS in tumor progression [11,21]. Our data suggest that these effects may be due to a reduction of enhanced Rac1 activity and cell migration noted after treatment with NAC in ATM-deficient cells (Fig. 4AeD). Accelerated migration in cells with high Rac1 activity may further contribute to the predisposition of cancer in the absence of functional ATM. Taken together, our results provide a missing link between ATM loss and the clinical features of the A-T such as an increase in both angiogenesis and cancer predisposition. Future studies examining the effects of antioxidant treatment in carriers of ATM mutations is warranted.
Acknowledgements The authors would like to thank Dr. Keith Burridge for helpful advice, Aaron Smith and Hannah Lee Dixon for technical assistance, and Dr. Erika Wittchen for critical review and editing of the manuscript. This work was funded by High Point University’s Research and Sponsored Programs (MCS), the Summer Undergraduate Research Program in the Sciences (MVB, MCS), and Undergraduate Research and Creative Works (MVB, MCS). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.033. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.033. References [1] Y. Shiloh, Y. Ziv, The ATM protein kinase: regulating the cellular response to genotoxic stress, and more, Nat. Rev. Mol. Cell Biol. 14 (2013) 197e210. [2] M. Choi, T. Kipps, R. Kurzrock, ATM mutations in cancer: therapeutic implications, Mol. Canc. Therapeut. 15 (2016) 1781e1791. [3] C. Barlow, P.A. Dennery, M.K. Shigenaga, M.A. Smith, J.D. Morrow, L.J. Roberts 2nd, A. Wynshaw-Boris, R.L. Levine, Loss of the ataxiatelangiectasia gene product causes oxidative damage in target organs, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 9915e9919. [4] Y. Peter, G. Rotman, J. Lotem, A. Elson, Y. Shiloh, Y. Groner, Elevated Cu/ZnSOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice, EMBO J. 20 (2001) 1538e1546.
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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[15] A. Aghajanian, E.S. Wittchen, S.L. Campbell, K. Burridge, Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif, PloS One 4 (2009) e8045. [16] G.A. Hobbs, L.E. Mitchell, M.E. Arrington, H.P. Gunawardena, M.J. DeCristo, R.F. Loeser, X. Chen, A.D. Cox, S.L. Campbell, Redox regulation of Rac1 by thiol oxidation, Free Radic. Biol. Med. 79 (2015) 237e250. [17] E.E. Sander, S. van Delft, J.P. ten Klooster, T. Reid, R.A. van der Kammen, F. Michiels, J.G. Collard, Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase, J. Cell Biol. 143 (1998) 1385e1398. [18] C.E. Tolbert, P.M. Thompson, R. Superfine, K. Burridge, S.L. Campbell, Phosphorylation at Y1065 in vinculin mediates actin bundling, cell spreading, and mechanical responses to force, Biochemistry 53 (2014) 5526e5536. [19] W.T. Arthur, K. Burridge, RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity, Mol. Biol. Cell 12 (2001) 2711e2720. [20] J.M. Atienza, N. Yu, S.L. Kirstein, B. Xi, X. Wang, X. Xu, Y.A. Abassi, Dynamic and label-free cell-based assays using the real-time cell electronic sensing system, Assay Drug Dev. Technol. 4 (2006) 597e607. [21] K. Ito, K. Takubo, F. Arai, H. Satoh, S. Matsuoka, M. Ohmura, K. Naka, M. Azuma, K. Miyamoto, K. Hosokawa, Y. Ikeda, T.W. Mak, T. Suda, A. Hirao, Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes, J. Immunol. 178 (2007) 103e110. [22] Y. Okuno, A. Nakamura-Ishizu, K. Otsu, T. Suda, Y. Kubota, Pathological neoangiogenesis depends on oxidative stress regulation by ATM, Nat. Med. 18 (2012) 1208e1216. [23] M. Nishikawa, Reactive oxygen species in tumor metastasis, Cancer Lett. 266 (2008) 53e59.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress Caitlin E. Tolbert a, Matthew V. Beck b, Claire E. Kilmer c, Melissa C. Srougi b, * a
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27514, USA Department of Chemistry, High Point University, High Point, NC, 27268, USA c Biotechnology Program, North Carolina State University, Raleigh, NC, 27607, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2018 Accepted 5 December 2018 Available online xxx
Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is integral in the response to DNA double-stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM-deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor-driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N-Acetyl-L-cysteine restores wild-type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro-migratory cellular behavior. © 2018 Elsevier Inc. All rights reserved.
Keywords: ATM Rac1 Reactive oxygen species Migration
1. Introduction Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by immunodeficiency, progressive cerebral ataxia, oculocutaneous telangiectasia, and a high incidence of cancer [1]. Individuals with A-T have ~30e40% cumulative incidence of cancer by age 40, primarily lymphoma and leukemia although breast, gastric, and other solid tumors also occur at high rates [2]. A-T is caused by a mutation in the A-T-mutated (ATM) gene. ATM is one of the most commonly mutated genes in lung adenocarcinoma, pancreatic cancers, and hematologic malignancies, while methylation or deletion of ATM has been reported in breast cancers. Functionally, ATM is a serine/threonine kinase related to the PI3Klike family of kinases. ATM is integral to the DNA damage response (DDR) pathway and is activated by double-stranded breaks (DSBs) such as after ionizing radiation (IR) exposure [3]. Upon activation by DSBs, ATM promotes DNA repair via cell cycle checkpoint activation.
* Corresponding author. Department of Chemistry, High Point University, One University Parkway, Couch Hall Rm 355D, High Point, NC, 27268, USA. E-mail address: [email protected] (M.C. Srougi).
Interestingly, A-T patients and ATM-/- cells exhibit high levels of oxidative stress, which may contribute to insulin resistance, neurodegeneration, and cancer progression observed in A-T [4,5]. A growing body of research suggest that mitochondrial dysfunction may contribute directly to elevated oxidative stress in A-T cells. These studies highlight a reduction in total mitochondrial respiratory activity, membrane potential (D4m), and expression of ROS detoxifying genes resulting in mitochondrial ROS overproduction and decreased ATP levels [6,7]. Given this context, ATM has a noncanonical role as a redox-sensor that maintains cellular redox homeostasis by modulation of antioxidant levels [8]. In ATM-null mice, several cancer types and neuromotor functions have improved with antioxidant treatment, which restore cellular redox homeostasis and D4m [9e11]. However, a direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Due to its role in regulating actin reorganization and cell migration, Rac1 GTPase is a critical component of tumorigenesis and invasiveness. Rac1 and other Rho GTPase family members are overexpressed in some human cancers however, unlike the related Ras GTPases, they are not activated by oncogenic mutations [12]. Instead, increased Rac1 activity may occur by altering the activity of
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Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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regulatory proteins. As with all canonical GTPases, activity is based on its nucleotide bound state: GTP-bound protein is “on” versus GDP-bound protein is “off”. Guanine nucleotide exchange factors (GEFs) facilitate the GTP-bound activated state, which signal to downstream effectors to alter cellular behavior. Besides regulatory proteins, some GTPases can be directly activated by ROS and reactive nitrogen species (RNS) in the absence of GEFs. Studies have identified a redox-sensitive motif in the phosphoryl-binding loop of Rho family members. Oxidation of reactive cysteines (Cys16/Cys20) within this motif destabilizes the bound nucleotide, allowing binding of GTP due to its excess in the cytosol. Treatment of recombinant GTPase with superoxide anion radical or nitrogen dioxide radicals in vitro induced guanine nucleotide dissociation, which could be reversed by radical quenching agents [13,14]. Further studies demonstrated direct activation of RhoA by ROS, causing actin cytoskeleton rearrangements [15]. Similar to RhoA, in vitro studies with Rac1 have shown that both ROS and RNS alter the charge of Cys18 leading to nucleotide exchange and subsequent activation of Rac1 [13,16]. Here, we report that cells lacking ATM kinase activity exhibit altered cytoskeletal dynamics, enhanced cell migration, and increased Rac1 activity. Loss of Rac1 in ATM-null cells decreased cell migration to levels similar to cells expressing wild-type ATM. Further investigation revealed that activation of Rac1 in ATM-null cells was due to enhanced ROS generation. Antioxidant treatment not only abrogated Rac1 activity but also reverted cell migration back to wild-type levels. These studies identify and highlight a role for Rac1 in promoting migration under conditions of elevated oxidative stress such as with ATM loss, which may facilitate tumorigenesis and metastasis noted in A-T patients and ATMmutated cancers. 2. Materials and methods 2.1. Reagents and antibodies CM-H2DCFDA was obtained from Molecular Probes (Eugene, OR). The Rac1 antibody (Clone 102), anti-Paxillin (Clone 165) were from BD Biosciences. Anti-GAPDH antibody was from SigmaAldrich (St. Louis, MO). Alexa Fluor 594 phalloidin and goat antimouse Alexa Fluor 488 were obtained from Molecular Probes. Peroxidase-conjugated goat anti-mouse and goat anti-rabbit were from Santa Cruz Biotechnology. Fibronectin (FN) was from Fisher Scientific.
camera and acquired using Metamorph Workstation (Universal Imaging Corp., META-40002). Cell morphology analysis was performed using NIH Image J software. Stress fiber (SF) formation was quantified by manual counting of cells containing at least one SF that transversed the cell. Cell circularity was defined as cell area divided by cell perimeter. Values ranged from 0 to 1.0 indicating an elongated or rounded morphology, respectfully. Actin-enriched ruffles were quanitifed from phalloidin stained cells. 2.4. Rac1 activity assays and western blotting Plated cells were washed with ice-cold PBS, lysed on ice, and assayed for Rac activation by glutathione S-transferase-p21activated kinase pulldown, as described by Sander et al. [17]. Bead fraction (active Rac) and total cell lysates (total Rac) were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and immunoblotted with the indicated primary and HRP-secondary antibodies. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and visualized using a BioRad ChemiDoc system. For quantification of western blots, intensity of bands from three separate experiments was obtained using Image J. 2.5. RTCA and cell spreading on fibronectin Assays were performed as previously described [18]. Briefly, cells were serum-starved for 3e16 h in high glucose DMEM/0.5% delipidated BSA (Sigma-Aldrich) prior to trypsinization and being held in suspension for 2 h in the same media. The xCELLigence RealTime Cell Analyzer (RTCA) system (Acea Biosciences/Roche Applied Science 05-469-759-001) was used to measure electrical impedance. Wells were coated with 50 mg/mL FN, then blocked with DMEM/0.5% delipidated BSA before seeding 2000 cells per well. The RTCA system uses electrical impedance signals, expressed as “cell index” (CI), to monitor cell adhesion and spreading of cells grown directly on microelectrode-coated surfaces. CI was recorded every 15 min for 4 h. Experiments were repeated three independent times. 2.6. ROS generation Formation of ROS was monitored by the conversion of nonfluorescent 5-(and-6)-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) to fluorescent DCF according to manufacturer's instructions (Invitrogen).
2.2. Cell culture GM16667 (ATMþ/þ cells) and GM16666 (ATM-/- cells) were from the Coriell Institute (Camden, NJ). HeLa and REF52 cells were from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in high glucose-containing DMEM with 10% fetal bovine serum (FBS), or 15% FBS (for ATM cells), 100 mg/mL hygromycin (for ATM cells), 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37 C in a 5% CO2 humidified atmosphere. All tissue culture components were from Invitrogen (Carlsbad, CA). KU55933 (KU) and N-Acetyl-L-cysteine (NAC) were from Sigma. 2.3. Immunofluorescence Coverslips were fixed in 3.7% paraformaldehyde and permeabilized in 0.2% Triton X-100/phosphate-buffered saline (PBS) prior to staining with indicated primary and secondary antibodies. Immunofluorescence images were taken with a Zeiss axiovert 200M microscope equipped with a Hamamatsu ORCA-ERAG digital
2.7. siRNA transfection HeLa cells at ~60% confluence were transfected with nontargeting siRNA (siControl) or siRNA targeted to Rac1 (siRac1) (Dharmacon) at 25 nM using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Wound healing assays were performed 48 h after transfection. 2.8. Wound healing assays Cells were plated on 30 mg/mL FN-coated coverslips and grown to confluency. Three parallel scratches were made on the cell monolayer using a 200 mL pipette tip, and media was replaced with 2% FBS-containing media. Where indicated, medium was supplemented with 10 mM KU55933, 0.5 mM NAC or DMSO. Live-cell images were taken of 4 random fields along the scratch at 0 and 5 h after wounding using an Olympus IX73 microscope (Olympus, Center Valley, PA) equipped with a CMOS 16 bit camera. Wound width was quantified using NIH Image J and represented as average
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% wound closure in 5 h from the initial width at 0 h. 2.9. Single-cell random migration For live single-cell tracking, cells were sparsely seeded onto 10 mg/ml FN-coated glass bottom culture dishes (MatTek Corporation) for 4e6 h prior to imaging. Time-lapse microscopy was performed with a Nikon BioStation IM (20x objective). Images were acquired every 10 min for 12 h. Cell velocity, persistence, and distance migrated was measured with NIH ImageJ using the Manual Tracking plug-in (http://rsbweb.nih.gov/ij/plugins/track/track. html). 3. Results 3.1. Loss of ATM kinase activity increases cellular spreading and Rac1 activation To determine if ATM activity alters cytoskeletal dynamics we first examined the spreading ability of ATM-inhibited cells. When cells are held in suspension for 2 h and then re-plated onto FN, coordinated control of actin cytoskeletal remodeling by several members of the Rho family of GTPases occurs. Cells change morphology from round to flattened with numerous matrix adhesions. These changes are attributed to the biphasic activation of RhoA and Rac1, with Rac1 promoting cell ruffling and spreading [19]. Due to tight control during cell spreading, this process provides a readout for perturbations in GTPase activity. REF52 cells were serum-starved and then held in suspension for 2 h while treated with the selective ATM kinase inhibitor KU55933 (KU). Once plated on FN, KU-treated cells displayed altered cytoskeletal rearrangement kinetics, displaying enhanced stress fibers (SFs) by 15 min compared to vehicle -treated cells (Fig. 1A). A significant decrease in cell circularity was noted in KU-treated cells suggesting increased radial spreading as cells begin to adhere (Fig. 1B). Increased SF formation and actin-enriched ruffles were also observed in KU-treated cells compared to vehicle-treated cells. (Fig. 1A and 1C-D). These observed changes in cytoskeletal dynamics during early spreading events suggested altered Rho GTPase activity. Therefore, we examined basal Rac1 activation in HeLa cells treated with KU. We observed that serum-starved HeLa cells pretreated with KU exhibited a significant 2-fold increase in active Rac1 compared to vehicle-treated cells (Fig. 1E). A significant 2-fold increase in active Rac1 was also confirmed in ATM-/- human fibroblasts derived from patients with A-T compared to an isogenic cell line re-expressing wild-type ATM (ATMþ/þ) (Fig. 1F). Furthermore, the increase in Rac1 activity was confirmed in HeLa cells transfected with a kinase-dead ATM construct (Supplementary Fig. 1). Together, these studies suggest that loss of ATM kinase function not only alters cytoskeletal dynamics, but also increases basal Rac1 activity. 3.2. Loss of ATM kinase activity increases cellular spreading and migration To evaluate the role of ATM in the long-term dynamics of cellular spreading and migration, we used the ATM-/- and ATMþ/þ human fibroblast model. Upon plating on FN, ATM-/- fibroblasts exhibited enhanced SF formation by 20 min. In contrast, ATMþ/þ cells showed SF formation beginning at 30 min (Supplementary Fig. 2), consistent with our previous findings (Fig. 1AeD). Rac1 activation remained high in ATM-/- cells from 0 to 20 min after adhesion, and dissipated by 30 min, the time when SF formation peaked (Fig. 2A and Supplementary Fig. 2). Conversely, ATMþ/þ cells showed characteristic biphasic activation of Rac1 where levels
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started high, decreased upon adhesion, and then increased again at 30 min (Fig. 2A) [19]. Since Rac1 is significantly activated upon loss of ATM kinase activity, we wanted to monitor the downstream effects on cellular spreading and migration. To do this, we used the automated RTCA (Real-Time Cell Analysis) system to quantify cell spreading [20]. ATMþ/þ and ATM-/- fibroblasts were plated at sub-confluency on FN-coated microelectrode E-Plates and impedance was analyzed over time. As shown in Fig. 2B (left panel), cell index (CI) was increased with ATM-/- cells compared to control ATMþ/þ cells, suggesting increased cell spreading. Loss of ATM significantly increased CI at both 2 h and 4 h compared to cells expressing wildtype ATM (Fig. 2B, right panel). We next evaluated the long-term effects of altered cytoskeletal dynamics on cell motility using time-lapse microscopy of ATMþ/þ and ATM-/- cells grown on FN. ATM-/- cells migrated significantly faster (0.6 mm/min ± S.D. 0.02) than ATMþ/þ cells (0.3 mm/ min ± S.D. 0.04) (Fig. 2C). ATM-/- cells also migrated significantly farther compared to ATMþ/þ cells, with a mean distance of ~130 mm (þ/ S.D. 32) versus 80 mm (þ/ S.D. 30), respectively (Fig. 2D). Together, these data suggest that loss of ATM results in Rac1 activation, increased cell spreading, and enhanced motility compared to wild-type cells. 3.3. Enhanced cell migration in ATM-inhibited cells requires Rac1 To explore the direct role of Rac1 in ATM-mediated alterations in cell migration, we knocked-down Rac1 in HeLa cells using siRNA and performed Rac1 activity and wound healing assays. HeLa cells were transfected with a non-targeting control RNAi (siControl) or RNAi targeted to Rac1 (siRac1), treated with DMSO or KU, and Rac1 activity was measured. Low Rac1 activity was observed in siControl DMSO-treated HeLa cells (Fig. 3A). In contrast, elevated Rac1 activity was detected in siControl KU-treated cells as previously observed (Fig. 1D). In siRac1 KU-treated cells, Rac1 activity was abrogated to a similar level as DMSO-treated siControl cells (Fig. 3A). Using these experimental conditions, we examined the effects of reduced Rac1 activity on cellular migration in ATMinhibited cells. Wound healing assays were performed on confluent monolayers 48 h post-transfection and the percent wound closure determined after 5 h. Migration was significantly increased in siControl KU-treated cells compared to siControl DMSO-treated (~41% versus ~23%, respectively) (Fig. 3B). This is consistent with increased Rac1 activity observed in KU-treated cells (Figs. 1E and 3A). However, siRac1 KU-treated cells exhibited a significant decrease in migration (~12%), compared to siControl KUtreated cells (Fig. 3B). Cells transfected with siRac1 and treated with DMSO had no significant changes in migration compared to siControl DMSO-treated cells (16% versus 23%, respectively). Collectively, these data suggest that Rac1 is important in the enhanced migratory phenotype noted in cells lacking ATM kinase activity. 3.4. Elevated ROS activates Rac1 in cells deficient in ATM kinase activity Previous data have shown that ATM-/- mice or A-T patients have higher levels of oxidative stress than wild-type and that this deleterious phenotype can be mitigated by treatment with the antioxidant NAC [4,21]. Furthermore, in vitro, Rho proteins can be directly activated by redox agents such as ROS and RNS [13,14]. We therefore hypothesized that loss of ATM would result in elevated ROS levels that could contribute to the activation of Rac1. We directly monitored intracellular ROS formation using the conversion of non-fluorescent CM-H2DCFDA to DCF in ATMþ/þ and ATM-/-
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 1. Inhibition of ATM kinase activity increases cell spreading and Rac1 activation. (A) Cells were plated onto FN-coated coverslips for the indicated times, then fixed and stained with phalloidin to visualize F-actin and with anti-paxillin to visualize focal adhesions. Bar, 40 mm. (B) Cell circularity (C) percentage of cells with F-actin and (D) average number of ruffles per cell were quantified for each treatment group in triplicate. Error bars indicate S.E.M. *p 0.01; **p 0.005; ***p 0.001. (E) Rac1 activity was measured by GST-PBD pulldown assay in HeLa cells treated with DMSO or KU, or (F) in ATM proficient (ATMþ/þ) or deficient (ATM-/-) human fibroblasts. For all graphs, average fold-change values from three independent experiments are represented ± S.E.M. *p 0.05.
cells. ATM-/-cells had significantly higher basal levels of ROS compared to ATMþ/þ cells (Fig. 4A). NAC treatment significantly decreased DCF fluorescence in ATM-/- cells to wild-type levels (þ/ NAC treatment) (Fig. 4A) [21], confirming that the increase in DCF fluorescence seen in ATM-/- cells was due to increased ROS generation. To determine if the elevated active Rac1 observed in ATM-/cells was due to elevated ROS, we examined Rac1 activity ± NAC. ATMþ/þ cells had low basal levels of active Rac1 with or without NAC treatment. However, the increase in active Rac1 seen in ATM-/-cells was prevented upon the addition of NAC (Fig. 4B). These data suggest that loss of ATM leads to increased susceptibility to oxidative stress, leading to Rac1 activation. Based on these findings, we postulated that antioxidant treatment should ameliorate Rac1-induced migration in ATM-/- cells. DMSO- and NAC-treated HeLa cells exhibited a wound closure of 10% and 11%, respectively, at 5 h (Fig. 4C and D). KU-treatment significantly increased wound closure (19%) compared to DMSOtreated cells (10%) (Fig. 4C and D). However, combined treatment with KU and NAC reverted wound closure to levels similar to control (7%) (Fig. 4D). Collectively, these data suggest that loss of ATM kinase activity enhances cell migration through the ROS-
mediated activation of Rac1. 4. Discussion Hereditary mutations of genes involved in DNA repair such as ATM markedly increase susceptibility to a variety of human cancers. Somatic mutations in the ATM gene are among the most commonly found aberrations and are associated with poor patient prognoses and chemotherapy resistance [2]. ATM functions not only in the maintenance of genomic integrity, but also cellular redox status. One mechanism for ATM's role in oxidative stress involves the regeneration of NADPH by promoting the pentose phosphate pathway, thus maintaining the active reduced forms of multiple detoxification proteins including thioredoxin 1, catalase, glutathione peroxidases, and superoxide dismutase [8,22]. Our data suggest that high basal Rac1 activity in cells lacking ATM kinase activity is linked to chronic oxidative stress. We demonstrate that loss of ATM kinase activity accelerates cellular spreading and migration (Fig. 1AeD and Fig. 2BeD). In ATM-/- or ATM-inhibited cells, elevated ROS levels result in increased steady-state Rac1GTP, which promoted enhanced cell motility (Fig. 4AeD). Down-
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 2. ATM deficiency enhances the kinetics of Rac1 activation, spreading, and migration upon adhesion to FN. ATMþ/þ and ATM-/- human fibroblasts were serum-starved and then held in suspension for 2 h. (A) Rac1 activity was determined upon adhesion to FN-coated dishes at indicated time points. Quantification of blots indicates the fold-change values from three independent experiments ± S.E.M. *p 0.05. (B) ATM loss enhances cellular spreading as determined by Real-time Cell Impedance Assay (RTCA). (Left panel) Representative trace of ATMþ/þ and ATM-/- human fibroblasts, graphed as Cell Index at 10 min intervals; lower Cell Index indicate decreased cellular spreading. Each data point is the average Cell Index of at least quadruplicate wells ± S.D. for each cell line. (Right panel) Graph showing relative Cell Index (cellular spreading) of ATMþ/þ and ATM-/- human fibroblasts at 2 h and 4 h. Data is the average ± S.D. combined from three independent experiments *p 0.01. (CeD) ATM deficiency increases cellular velocity and distance migrated. Singlecell live imaging was performed on ATMþ/þ and ATM-/- cells after adhesion to FN for 24 h to track (C) velocity and (D) distance traveled. Graphs represent cell averages per experiment (n ¼ 30) þ/ S.D., *p 0.05.
Fig. 3. Knock-down of Rac1 suppresses enhanced cell migration in ATM-inhibited cells. HeLa cells were transfected with a non-targeting siRNA oligo (siControl) or siRNA oligos targeted to Rac1 (siRac1). Cells were then treated with vehicle (DMSO) or KU. (A) Rac1 activity was assessed by GST-PBD pulldown assay. (B) Wound healing assay. Confluent cell monolayers were scratched with a micropipette tip. The average percent wound closure after 5 h was calculated from three independent experiments. *p 0.01, **p 0.001, not significant (NS).
regulation of active Rac1 using siRNA or the free radical scavenger NAC, reverted cells back to a reduced migration phenotype similar to wild-type cells (Figs. 3B and 4D). These data demonstrate for the first time a physiological linkage between ATM status and the regulation of Rac1 activity, which could have important therapeutic implications especially in human cancers.
Work by Heo et al. [13,14] demonstrated that ROS and RNS can directly activate Rho GTPase family members, in the absence of GEFs. In this context, activation of the GTPase can be reverted by a reducing environment or the presence of a redox scavenger (Fig. 4B). Based on our findings, we speculate that in cases of increased physiological oxidative stress, such as occurs with ATM
Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Fig. 4. Increased ROS in ATM-deficient cells activates Rac1 and increases cell migration. Confluent monolayers of ATMþ/þ and ATM-/- human fibroblasts were loaded with CMH2DCFDA and then treated ± the free radical scavenger N-Acetyl-L-cysteine (NAC). Generation of ROS was monitored by the fluorescence intensity of DCF. Graph represents average from quadruplicate wells of n ¼ 3 independent experiments ± S.E.M; *p 0.05. NAC blocks Rac1 activation and enhanced migration in cells lacking ATM kinase activity. (B) (Left panel) ATMþ/þ and ATM-/- cells were treated ± NAC and Rac1 activity was assessed using GST-PBD pulldown assay. (Right panel) Fold-change in active Rac1, where the graph represents the average from three independent experiments ± S.E.M.; *p 0.05. (C) Phase contrast images of HeLa cell monolayers treated with DMSO, KU, NAC, or both NAC and KU at 0 and 5 h after wounding. (D) Quantification of the average percent wound closure after 5 h was calculated from three independent experiments; *p 0.01, **p 0.001.
loss, ROS-mediated regulation of Rho GTPases may dominate. However, we do not discount that parallel GEF-independent and -dependent pathways may be involved in an ATM-deficient/ mutated background. Therefore, ongoing research will investigate the contribution of GEF-mediated activation of Rac1 under physiological oxidative stress. Clinically, ROS may increase tumor metastasis by promoting epithelial-mesenchymal transition, migration and invasion of tumor cells, and angiogenesis [23]. Rho family GTPases are also implicated in the invasiveness of various cancers. Elevated Rac1 activity in this context would support cancer metastasis and vascularization by promoting directed migration (Fig. 4C and D). Not surprisingly, carriers of biallelic mutations responsible for A-T have a two-fold higher risk of developing cancer, especially breast cancer, compared to monoallelic mutation carriers. Furthermore, down-regulation of ATM expression in somatic breast carcinomas is correlated with increased microvascularization [11]. Our data uncover an important pathophysiological role for ROS-induced Rac1 activity in the absence of functional ATM. Previous findings have shown the benefits of NAC treatment in ATM-deficient model systems to counteract the effects of ROS in tumor progression [11,21]. Our data suggest that these effects may be due to a reduction of enhanced Rac1 activity and cell migration noted after treatment with NAC in ATM-deficient cells (Fig. 4AeD). Accelerated migration in cells with high Rac1 activity may further contribute to the predisposition of cancer in the absence of functional ATM. Taken together, our results provide a missing link between ATM loss and the clinical features of the A-T such as an increase in both angiogenesis and cancer predisposition. Future studies examining the effects of antioxidant treatment in carriers of ATM mutations is warranted.
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Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033
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Please cite this article as: C.E. Tolbert et al., Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.033