ATF3 suppresses mutant p53 function

Activating transcription factor 3 suppresses the oncogenic function of mutant p53

Saisai Wei 1,6, Hongbo Wang 1,4, Chunwan Lu 2, Sarah Malmut 1, Jianqiao Zhang 4, Shumei Ren1, Guohua Yu 5, Wei Wang 5, Dale D. Tang6, and Chunhong Yan1,2,3

1 Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208; 2 GRU Cancer Center, 3 Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, GA 30912; 4 Key Laboratory of Molecular Pharmacology and Drug Evaluation, School of Pharmacy, Yantai University, Yantai 264005, China; 5 Department of Pathology, Affiliated

Yantai Yuhuangding Hospital, Medical College of Qingdao University, Yantai 264000, China; 6 Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208.

* Running Title: ATF3 suppresses mutant p53 function To whom correspondence should be addressed: Chunhong Yan, GRU Cancer Center, 1410 Laney Walker Blvd., CN-2134, Augusta, GA 30912 . Tel: 706-721-0099; E-mail: cyan@gru.edu Key Words: mutant p53; ATF3; drug resistance; migration; invasion; lung cancer

Background: Mutant p53 often acquires tumor-promoting activities including promoting cancer cell survival, invasion and metastasis. Results: Activating transcription factor 3 (ATF3) bound mutant p53 proteins, sensitized cancer cells to chemotherapy, and suppressed mutant p53-mediated cell migration. Conclusion: ATF3 suppressed the tumor-promoting function of mutant p53. Significance: Targeting ATF3 could be a novel strategy for treating p53-mutated cancer. ABSTRACT Mutant p53 proteins (mutp53) often acquire oncogenic activities conferring drug resistance and/or promoting cancer cell migration and invasion. Whereas it has been well established that such gain-of-function is mainly achieved through interacting with transcriptional regulators thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g., R175H and R273H) and subsequently suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53.

Accordingly, ATF3 promoted expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and thus lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that downregulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function thereby contributing to tumor suppression in TP53-mutated cancer.

The tumor suppressor p53 gene (TP53) is one of the most frequently mutated genes in human cancers. Found in about a half of human cancer, TP53 mutations frequently occur in residues residing in the central DNA-binding domain. These “hot-spot” mutations (e.g., R175H, R273H) lose the ability to regulate expression of canonical p53 target genes and thereby are unable to suppress tumorigenesis caused by various oncogenic stimuli (1). In addition to the loss of tumor suppressor activity, mutant p53 proteins (mutp53) often acquire oncogenic activities including promoting cell proliferation, conferring drug resistance, inducing angiogenesis, and most importantly, promoting cancer cell invasion and metastasis (1). Indeed, whereas p53 mutation is often associated with poor prognosis of cancer, mice carrying hot-spot p53 mutations develop

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http://www.jbc.org/cgi/doi/10.1074/jbc.M113.503755The latest version is at JBC Papers in Press. Published on February 19, 2014 as Manuscript M113.503755

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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aggressive tumors characterized by a high frequency of metastasis (2,3). Although mutp53 may directly regulate gene expression by binding to DNA elements distinct from the wild-type p53 (4), these mutated proteins more often achieve their gain-of-function through binding to transcriptional regulators (e.g., NF-Y, p63) thereby indirectly altering transcription (4,5). p63 is a member of the p53 family of transcription factors and can regulate expression of genes (e.g., SHARP1) involved in migration and invasion (6-8). p63 also controls cell migration and invasion by downregulating integrin recycling (9). A subset of common p53 mutants, but not the wild-type p53, can efficiently bind and inactivate p63 thereby promoting cancer cells to invade and metastasize (9,10). Relevant to our research interests, mutp53 was shown to suppress phorbol ester-induced expression of activating transcription factor 3 (ATF3) and prevent ATF3-mediated cell death (11). ATF3 is a member of the ATF/CREB family of transcription factors, and can bind to the ATF/CREB cis-regulatory element and regulate gene expression under various conditions. Although ATF3 is aberrantly expressed in a wide range of human cancer (12), its role in cancer remains unclear. Whereas it was shown to promote tumorigenesis and metastasis in skin, breast and prostate cancer (13-15), ATF3 was also found to suppress oncogenic networks, induce cell death, and inhibit malignant dissemination in many cancer types including glioblastoma, colon, prostate, bladder and lung cancer (16-20). Such a context-dependent role that ATF3 plays in cancer is likely due to complex protein-protein interaction networks that ATF3 is involved. Indeed, in addition to transcriptional regulation, ATF3 has been found to interact with many critical cellular proteins and regulate their functions. One of the well-characterized ATF3-binding proteins is the wild-type p53 (21,22). ATF3 binds the p53 C-terminus via its leucine zipper domain (ZIP), and this interaction prevents p53 from ubiquitin-mediated degradation thereby activating p53 in response to DNA damage (23). Interestingly, ATF3 also binds the major p53 suppressor MDM2, and serves as a bona fide substrate for the E3 ubiquitin ligase (24). Given that MDM2 is a p53 target gene, the ATF3-p53-MDM2 interplay likely fine-tunes p53 tumor suppressor activity in response to oncogenic challenges. ATF3 also

binds human papillomavirus (HPV) E6 protein and activates p53 in HPV-positive cancer cells by blocking p53 ubiquitination (25). Moreover, ATF3 represses androgen signaling by binding to the androgen receptor in prostate cancer (26). These findings support a notion that ATF3 can regulate cancer development and progression via protein-protein interaction. In line with this notion, we report here that ATF3 bound mutp53 to reverse drug resistant and suppress migration and invasion of p53-mutated cancer cells. EXPERIMENTAL PROCEDURES Cell Culture, Plasmids, and Transfections - H1229 and MDA-MB-231 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, while SKBR3 cells were cultured in McCoy’s 5a medium. These cells are routinely maintained in our laboratory. The A431 cell line, cultured in DMEM medium, was a kind gift from Dr. Shi-Yong Sun. We obtained plasmids expressing the wildtype p53, R175H, R273H, R249S and V143A from Dr. Bert Vogelstein. The coding sequences of these proteins were amplified by PCR and cloned into pGEX-3X to express GST fusion protein in bacteria. The construct expressing FLAG-p63 was obtained from Addgene (Cambridge, MA). Plasmids expressing ATF3 and ∆ATF3 were described previously (23,26). These genes were cloned into pIRES2-GFP (BD Biosciences) for bicistronic expression of ATF3 with GFP. Transfections were carried out using Lipofectamine 2000 according to the manufacturer’s protocol. FACSAria II Flow Cytometer (BD Biosciences) was used to sort GFP-positive cells for gene expression analysis. In Vitro Translation and GST-pulldown Assays –The TNT Quick Coupled Transcription /Translation System (Promega) were used for in vitro translation of ATF3 and ∆ATF3. Briefly, 1 µg of plasmids were incubated with 40 µl of rabbit reticulocytes lysates supplemented with 1 µl of 20 µM methionine at 30 °C for 90 min. In vitro-translated proteins were then incubated with GST fusions immobilized onto glutathione agarose at 4°C overnight. After extensive washes, bound proteins were resolved in SDS-polyacrylamide gels and detected by immunoblotting as described previously (26). shRNA Knockdown and Retroviral Infections - p53 and ATF3 knockdown was performed using a Lentivector-based shRNA system (pSIH-H1

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shRNA Cloning and Lentivector Expression system, System Biosciences). The p53-targeted sequence was 5’-GAC TCC AGT GGT AAT CTA C-3’, based on a publication (27). The ATF3 targeted sequences were reported previously (25). For negative controls, a luciferase-targeted sequence (5’-CTT ACG CTG AGT ACT TCG A-3’) was cloned into the Lentivector. For retroviral-mediated gene transfer, the ATF3-coding sequence was cloned into pBabe, and then transfected into Ampho293 (Clontech) to pack retrovirions for infections as described (23). Immunoblotting and Co-immunoprecipitations (co-IP) assays - Immunoblotting was carried out as described previously (28). For co-IP assays, transfected cells were lysed in RIPA lysis buffer (10 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1 mM EDTA, 0.2% TritonX-100, 0.2 mM DTT, 10% glycerol, and protease inhibitors). Cell lysates (1 mg) were incubated with 1 µg of the ATF3 antibody or IgG at 4 °C overnight. Immunocomplex was precipitated with 25 µl of protein A agarose (Roche), washed with 1 ml of RIPA buffer supplemented with 300 mM NaCl for 3 times, and detected by immunoblotting. For FLAG-p63 IP, cell lysates were incubated with 25 µl of anti-FLAG M2 Affinity Gel (Sigma). Antibodies for p53 (DO-1), ATF3 (C-19), and p63 (4A4) were purchased from Santa Cruz Biotechnology. The β-actin antibody were obtained from Sigma. Scratch-Wound Migration Assays and Time-lapse Imaging –This was performed according to a recent report (9). Briefly, cells were grown in monolayers, wounded with a pipette tip, and then grown in 1% FBS. The movement of individual cells into the wound was recorded with a phase contrast 20× objective using an inverted microscope (Olympus IX70). Images were obtained every 10 min for a total 16 hr. 40 to 60 cells were then tracked and analyzed for differences in speed and net distance using Image J. Transwell Migration and Invasion Assays - For Transwell migration assays, the lower surface of polycarbonate membranes (8 μm pore size, Corning Incorporation) were coated with 100 μg of fibronectin. Cells (2-5×105) suspended in 0.1 ml of medium (containing 0.1% BSA) were then dispensed into upper chambers of Transwells and incubated for 5 hrs. Cells migrated to the lower membrane surface were fixed, stained with

hematoxylin/eosin (Fisher Scientific), and counted under a microscope. For invasion assays, 106 cells were plated in Transwells coated with Matrigel and cultured for 24 hr. Invaded cells were stained and counted as described (29). Colony Formation Assays – 200, 1.5×104, or 2.5×104 cells plated in 6-well plates were treated with DMSO, 1.5 μM of etoposide for 48 hrs, or 2 μg/mL cisplatin for 24 hrs, respectively. After treatments, cells were washed and grown in normal culture medium for 9 days for colony formation. Colonies were stained with crystal violet and counted as described previously (25). Real-time RT-PCR – Total RNA was prepared, reverse transcribed and subjected to real-time PCR assays as described previously (30). The primer sequences used for real-time PCR are available upon request. Immunohistochemical Staining - Tumor samples were obtained from 36 lung cancer patients with radical resection in the Affiliated Yantai Yuhuangding Hospital, Medical College of Qingdao University, Yantai, China. All experimental procedures were approved by the Institutional Review Board of the Affiliated Yantai Yuhuangding Hospital, and written informed consents were obtained for all patient samples used in this study. Standard immunohistochemical procedures were carried out using anti-ATF3 antibody (HPA001562, 1:200, Sigma) and anti-p53 antibody (sc-6243, 1:600, Santa Cruz) as described previously (26). Briefly, 5 µm sections were incubated in hot citrate buffer to retrieve antigens, and then blocked in 5% normal goat serum. After incubation with primary antibodies overnight, sections were stained using the ABC Elite Kit and the DAB Kit (Vector) according to the manufacturer’s recommendations. The specimens were independently scored by 2 pathologists based on the staining intensity and subcellular localization (0, no staining; 1, weak staining; 2, strong staining in both cytoplasm and nucleus; and 3, strong nuclear staining). Statistic analyses were carried out using the Fisher’s Exact test or the Mann-Whitney test as indicated in Figure legends. RESULTS

ATF3 Interacts with Mutp53 - We previously showed that ATF3 binds the wild-type p53 at its C-terminus (aa363-393) (23) (Fig 1A). Since most p53 mutations occur in the DNA-binding domain

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(DBD), we reasoned that ATF3 could bind mutp53 proteins and affect their oncogenic activities. To test this, we carried out GST-pulldown experiments to examine binding of ATF3 to GST-fused hotspot p53 mutants including R175H, R273H, R249S and V143A. The GST-mutp53 fusion proteins, but not GST, pulled down in vitro-translated ATF3 (Fig 1B, lanes 4-7 vs. lane 2), indicating that ATF3 indeed interacted with these mutp53 proteins. ATF3 bound mutp53 at an affinity similar to that for its binding to the wild-type p53 since the amount of ATF3 down-pulled by mutp53 was comparable to that by the wild-type p53 (Fig 1B, lanes 4-7 vs. lane 3). To determine whether ATF3 also interacts with mutp53 in vivo, we expressed ATF3 and mutp53 in p53-null H1299 cells, and then precipitated ATF3 from cell lysates using an ATF3 antibody. The ATF3 antibody precipitated R175H, R273H, R249S, and V143A along with ATF3 (Fig 1C). Moreover, we carried out co-IP assays in R175H-harboring SKBR3 and R273H-expressing A431 cells to test whether endogenous ATF3 and mutp53 proteins interacts. The ATF3 antibody or the p53 antibody could co-immunoprecipitate both ATF3 and mutp53 proteins (Fig 1D, lane 3 vs. lane 2, lane 7 vs. lane 6), demonstrating that ATF3 can bind mutp53 in cells.

ATF3 Represses Mutp53-induced NFKB2 Expression and Reverse Drug Resistance - Given that ATF3 could bind mup53, we tested whether ATF3 affects the gain-of-function of mutp53. Towards this end, we transfected H1299 cells and developed cell lines expressing two most common mutp53 proteins - R175H and R273H. An empty vector cell line (V) was also developed and used as a control. The mutp53 expression level in these cells was comparable to that in A431 and MDA-MB-468 cells carrying native TP53 mutations (Fig 2A, lanes 2-3 vs. lanes 4-5). Since H1299 cells express a low level of ATF3, we overexpressed ATF3 in these mutp53 cell lines by retroviral infections, generating cells expressing mutp53 with or without ATF3 overexpression (Fig 2B). Not surprisingly, neither mutp53 nor ATF3 expression altered cell growth (Fig 2C). It was reported that mutp53 (e.g., R175H) induces NFKB2 expression to activate pro-survival NFκB signaling, thereby conferring drug resistance to cancer cells (31). Indeed, we found that the NFKB2 mRNA level and the NFκB transcriptional activity (represented by NFκB

luciferase reporter activity) were increased in the R175H-expressing cells (Fig 2D and 2E). Interestingly, ATF3 expression decreased the NFKB2 expression level and down-regulated the NFκB activity in the R175H cells (Fig 2D and 2E). Transient expression of ATF3 in the R175H cells was also sufficient to down-regulated NFκB transcriptional activity induced by the mutant protein (Fig 2F). These results suggest that ATF3 might reverse mutp53-mediated drug resistance. To test this, we treated cells with cisplatin or etoposide and then determined cell survival using colony formation assays. Whereas the R175H cells survived much better than the empty vector cells, ATF3 expression significantly decreased the number of viable R175H cells (Fig 2G and 2H). ATF3 did not alter the mutp53 expression level under these conditions (Fig 2I), suggesting that the ATF3-mediated inhibition of cell survival was rather a consequence of altered mutp53 activity. Of note, although it exerts pro-apoptotic activity under certain conditions (11, 17), ATF3 did not affect survival of H1299 cells without R175H expression (Fig 2G and 2H). In line with these results, knockdown of ATF3 expression using shRNA increased the number of survived R175H cells after the etoposide treatment (Fig 2J, left panel). Similarly, siRNA-mediated knockdown of ATF3 expression in SKBR3 cells carrying the endogenous R175H protein promoted cell resistance to etoposide treatments. An early report has demonstrated that resistance of SKBR3 cells to chemotherapeutic agents (including etoposide) were due to expression of the mutant p53 protein (32). Therefore, our results indicate that ATF3 can reverse drug resistance in p53-mutated cancer cells.

ATF3 Suppresses Mutp53-driven Cell Migration and Invasion - Mutp53 can promote cancer progression by driving cell migration and invasion. We therefore examine cell migration in scratch-wound assays using time-lapse microscopy (Fig 3A). Consistent with the migration-promoting activity of mutp53 (9), both R175H- and R273H-expressing H1299 cells moved significantly faster than the control cells evidenced by the significantly increased migration speed (Fig 2B). The mutp53 cells appeared to move more erratically into the wound (Fig 3A) and the net moving distance was significantly longer than that of the control cells (Fig 2C). Interestingly, ATF3 appeared to counteract the mutp53 activity and thus ATF3 expression in both R175H and R273H

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cells significantly decreased the migration speed and the net distance (Fig 3B and 3C). ATF3 expression also increased the persistence of the mutp53 cells and changed the cell migration tracks to a pattern similar to that of the control empty vector cells (Fig 3A). These results indicated that ATF3 suppressed mutp53-driven cell migration. To corroborate this finding, we carried out Transwell assays to examine cell migration driven by fibronectin – a mutp53-mediated event (9). Whereas R175H and R273H increased the number of cells migrated through membranes, ATF3 significantly suppressed migration induced by mutp53 (Fig 3D and 3E). It is important to note that ATF3 had minimal effect on migration of cells without mutp53 expression (Fig 3A-E). Therefore, ATF3 appeared to counteract mutp53 and suppress cell migration mediated by the mutant proteins. Consistent with these results, ATF3 expression suppressed invasion of the R175H cells through Matrigel (Fig 3F).

ATF3 Suppresses Migration in Cancer Cells Harboring TP53 Mutations - To test whether ATF3 affects function of native mutp53, we determined whether ATF3 also suppresses migration in A431 cells harboring a native R273H mutation. The migration of A431 cells was largely driven by the native R273H protein as knockdown of its expression (Fig 4A) dramatically decreased cell motility (Fig 4B). ATF3 expression indeed significantly decreased the migration speed in A431 cells (Fig 4B). In contrast, knockdown of ATF3 expression in these cells (Fig 4C) increased cell motility (Fig 4D). These results support a notion that ATF3 counteracts native mutant p53 proteins and suppresses their migration-promoting activity as well. The reason why ATF3 increased migration of the p53 knockdown cells (Fig 4B) is unknown, but might reflect the context-dependent nature of ATF3 function in cancer. We also determined effects of ATF3 on migration of MDA-MB-231 cells carrying the native R280K mutation. ATF3 similarly suppressed migration in this cell line, although the inhibition was less effective as compared to A431 cells (Fig 4F). This was probably because the mutp53 was not the major driving force for cell migration in MDA-MB-231 cells. Indeed, knockdown of p53 expression in MDA-MB-231 cells (Fig 4E) only modestly decreased cell migration (Fig 4F). Nevertheless, our results demonstrated that ATF3

can suppress migration of cancer cells carrying native TP53 mutations.

ATF3 Suppresses the Mutp53-p63 Interaction and Reactivates p63 in Mutp53 cells- A well-characterized mechanism by which mutp53 promotes migration and invasion is related to the interaction between mutp53 and p63, which can inactivates p63 transcriptional activity by preventing p63 from binding to target genes (4). Indeed, it has recently been demonstrated that a mutp53-regualted, p63-target gene SHARP1 is a major migration and metastasis suppressor (7,8). To explore the mechanism underlying the suppression of mutp53-driven migration by ATF3, we determined effect of ATF3 on the mutp53-p63 interaction. We co-expressed FLAG-p63, R175H/R273H, and ATF3 in H1299 cells, and carried out co-IP assays. The FLAG antibody efficiently co-precipitated R175H and p63 (Fig 5A, lane 2), but precipitated only a small amount of R273H (Fig 5A, lane 5), consistent with a previous report (9). These results confirmed the interaction between mutp53 and p63. ATF3 expression dramatically decreased the amounts of both R175H and R273H co-precipitated by FLAG-p63 (Fig 5A, lane 3 vs. lane 2, lane 6 vs. lane 5), indicating that ATF3 prevented mutant p53 from binding to p63. Interestingly, ATF3 was also found in the immunocomplexes, probably due to its interaction with R175H/R273H. Alternatively, ATF3 might directly bind p63. Indeed, whereas p63 was co-precipitated with ATF3 (Fig 5B, lane 5), ATF3 appeared to directly bind p63 as revealed by GST-pulldown assays (Fig 5C, lane 3). Given that ATF3 disrupted the mutp53-p63 interaction, we determined whether ATF3 affects the p63 transcriptional activity in mutp53- expressing cells. Using a luciferase reporter driven by a synthetic p63-responsive promoter, we found that the p63 transcriptional activity was repressed by R175H or R273H as expected (Fig 5D). ATF3 expression in these mutp53-expressing cells significantly increased the p63 transcriptional activity (Fig 5D), arguing for the notion that ATF3 can counteract mutp53 and reactivate p63 in cells harboring mutp53. In line with these results, whereas R175H or R273H repressed SHARP1 expression, ATF3 significantly increased the mRNA level of this p63-target gene in the mutp53-expressing cells (Fig 5E). The increase in the p63 reporter activity by ATF3 in the absence of R175H (Fig 5D) might be due to direct binding

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of ATF3 to p63. However, ATF3 did not appear to affect SHARP1 expression in the control cells (Fig 5E).

An ATF3 Mutant Deficient in Mutp53 Binding Fails to Suppress Mutp53-mediated Migration and p63 Inactivation - Since ATF3 can bind mutp53, we reasoned that this interaction could account for the suppression rendered by ATF3 on the mutp53 function. To test this, we took use of an ATF3 mutant (∆ATF3) devoid of the ZIP domain (aa102-139) known to mediate ATF3 binding to the wild-type p53 (23). This ATF3 mutant failed to bind R175H or R273H (Fig 6A, lanes 8 and 9), but retained the capability of binding p63 (Fig 6B, lane 3). We expressed ATF3 and ∆ATF3 bicistronically with GFP in the R175H cells (Fig 6C) and examined migration of ∆ATF3-expressing (GFP-positive) and -unexpressing (GFP-negative) cells. Consistent with the previous results (Fig 3), ATF3 expression dramatically suppressed migration of the R175H cells (Fig 6D and 6E, comparing R175H ATF3+ and R175H ATF3-). In contrast, ∆ATF3 did not significantly alter the net migration distance of the mutp53 cells while decreasing the migration speed to an extent significantly smaller than wild-type ATF3 (Fig 6D and 6E, comparing R175H ∆ATF3+ and R175H ∆ATF3-). Since ∆ATF3 failed to bind mutp53 but still bound p63, these results suggest that the ATF3-mtp53 binding is required for the suppression of mutp53-mediated migration by ATF3. The residual migration-inhibitory activity of ∆ATF3 might be due to dimerization of ∆ATF3 with the endogenous wild-type ATF3 (23), which might result in binding of a small amount of ∆ATF3 to R175H in cells. Of note, ∆ATF3 failed to counteract R175H-driven resistance to etoposide treatments as well (Fig 6F), supporting the notion that ATF3 suppresses mutp53 function through interacting with mutp53. We next carried out co-IP assays to determine whether ∆ATF3 lost the ability to disrupt the mutp53-p63 interaction. Indeed, the mutant ATF3 failed to prevent mutp53 from binding to p63 (Fig 6G, lane 4 vs. lane 3). Consistent with these results, ∆ATF3 failed to increase the p63 transcriptional activity repressed by R175H (Fig 6H). We also sorted ∆ATF3-expressing (GFP-positive) cells for QPCR to quantify the SHARP1 expression level. In contract to the effects of ATF3, ∆ATF3 failed to induce SHARP1 expression in the R175H cells (Fig 6I). These results suggest that the ATF3

mutant deficient in mutp53 binding lost the ability to reactivate p63 transcriptional activity in mutp53-expressing cells. Taken together, our results indicate that the binding of ATF3 to mutp53 reactivates p63 and accounts for the suppression of mutp53 oncogenic activities by ATF3.

ATF3 Expression Negatively Correlates with Metastasis of TP53 Mutated Lung Cancer - Given that ATF3 suppressed mutp53-mediated migration and invasion, we carried out immunohistochemical staining in human lung cancer samples to determine whether ATF3 expression correlates with metastasis in human cancer. We also stained the tissues with an anti-p53 antibody and used p53 immunopositivity as a surrogate marker for TP53 mutations (33). Both the ATF3 and p53 antibody generated clear staining on tumor sections (Fig 7A and 7B). Interestingly, neither ATF3 expression nor p53 mutation correlated with lymph node mestastasis in these lung tumors (Fig 7C). However, when we examined ATF3 expression in p53-positive samples, we found a significant inverse correlation between ATF3 expression and metastasis (Fig 7C). Whereas ATF3 positive staining was frequently found in p53-positive tumors free of lymph node metastasis, fewer metastatic, p53-positive tumors expressed ATF3 (Fig 7C). Such a correlation was not found in p53-negative tumors (Fig 7C). Further analysis by scoring ATF3 staining intensity (Fig 7D) (20) revealed that high ATF3 staining scores were often found in p53-positive tumors lacking lymph node metastasis (Fig 7E). These results are consistent with the notion that ATF3 counteracts mutp53 and suppresses metastasis in TP53-mutatd lung cancer. DISCUSSION The fact that TP53 mutations often correlate with poor prognosis of cancer underscores the importance of unveiling the mechanisms by which the mutp53 oncogenic function is regulated. In this study, we found that ATF3 bound and counteracted common mutp53 proteins thereby reversing drug resistance and suppressing migration of TP53-mutated cancer cells. Since ATF3 binds to the p53 C-terminus (23) often unaffected by TP53 mutations, it is likely that ATF3 could bind a majority of mutp53 proteins and regulate their oncogenic function. Our

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findings thus argue for that ATF3 is a novel suppressor of mutp53. To our knowledge, ATF3 is the only protein other than ANKRD1 (34) that has been demonstrated to suppress the oncogenic function of mutp53. Several other mutp53-binding proteins (e.g., PLK2, TopBP1) rather promote the mutp53 gain-of-function (35,36). This mutp53-suppressing activity is supported by our observations that ATF3 expression was lost or downregulated in metastatic lung cancer harboring TP53 mutations (Fig 7). Whereas the mechanism(s) underlying ATF3 loss/downregulation in these tumors is currently unknown, our results suggest that increasing ATF3 expression and/or enhancing the ATF3-mutp53 interaction through therapeutic approaches would benefit patients with TP53 mutated, metastatic cancer. Indeed, enforced expression of ATF3 in TP53-mutated cancer cell lines A431 and MDA-MB-231 suppressed malignant cell migration (Fig 3). Interestingly, ATF3 expression appeared to be downregulated by R175H and R273H in response to etoposide and cisplatin treatments (Fig 2I, lane 8 vs. lane 6, and lane 12 s. lane 10) – a reminiscence of a previous finding that mutp53 repressed ATF3 expression upon phorbol ester treatment (11). Given that ATF3 is a mutp53 suppressor, mutp53-mediated down-regulation of ATF3 expression might serve as a mechanism to ensure maximal oncogenic activity achieved by mutp53 under these conditions. This mutual regulation further highlights therapeutic values of targeting ATF3 and related cell signaling in treating TP53-mutated cancer. ATF3 can increase the stability of the wild-type p53 by preventing it from MDM2-mediated ubiquitination and degradation (23). However, ATF3 did not appear to affect mutp53 expression in the tested cells (Fig 2B, 4A, and 4E). This result is consistent with the previous reports that MDM2 promotes mutp53 degradation only in specific contexts (e.g., precancerous situations prior to oncogene activation) (37,38). Rather than altering the protein stability, our results argue for that ATF3 directly regulated the mutp53 oncogenic activity by binding to mutp53. The ATF3 binding prevented mutp53 from interacting with p63, resulted in reactivation of p63 and expression of anti-invasive p63 target genes (e.g., SHARP1) in mutp53-expressing cells. Since ATF3 binds to the p53 C-terminus distinct from the region where p63 binds (10), it is unlikely that the disruption of the

mutp53-p63 interaction by ATF3 is due to steric hindrance. More likely, the ATF3 binding alters the mutp53 conformation required for p63 binding (10). Interestingly, although it only weakly interacted with p63 (Fig 5A), R273H inactivated p63 and repressed SHARP1 expression as efficiently as R175H (Fig 2D). These results are consistent with the notion that the mutp53 C-terminus can inactivate p63 independent of its binding to p63 (9), suggesting that the interaction of ATF3 with the mutp53 C-terminus might directly reactivate p63 as well. It is important to note that ATF3 also appeared to bind p63 (Fig 5B and 5C). However, it was the ATF3-mutp53 interaction rather than the ATF3-p63 interaction that reactivated p63 as the ATF3 mutant (∆ATF3) that can bind p63 but not mutp53 failed to increase the p63 activity (Fig 6H). Consistent with an early report (39), we also found that ATF3 interacted with p73 (data now shown), the other p53 family member bound by mutp53 (10). Whether ATF3 interferes with the mutp53-p73 interaction to regulate mutp53 function remains elusive. Unlike p63, however, the role of p73 in cancer development and progression has not been well established. In addition to p63 targets, we found that ATF3 also regulated expression of other mutp53 targets including NFKB2 (Fig 2D). However, ATF3 did not appear to regulate NFKB2 expression in the cells that did not carry mutp53 (Fig 2D), arguing against the possibility that the ATF3 transcriptional activity caused the NFKB2 downregulation in mutp53-expressing cells. Rather, the binding of ATF3 to mutp53 might alter the interactions of mutp53 with other transcriptional regulators (e.g., VDR and ETS2) (40,41), or DNA elements (5), thereby regulating gene expression. These could be achieved through conformational changes or steric hindrance. Given that mutp53 interacts with proteins or DNA through varying mechanisms (5), ATF3 might affect a subset of mutp53 target genes, but leave other mutp53 targets unaffected. Indeed, we found that ATF3 also repressed hTERT expression, but not ITAG6 or E2F expression, in the R175H cells (data not shown). Expression of other reported mutp53 target genes including EBAG9, survivin, cdc25A and CXCL1 (5) was not altered by R175H in our H1299 cells (data not shown), suggesting that the cell context might contribute to gene expression regulated by mutp53.

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ATF3 was identified as a metastasis promoter in an early study investigating the metastatic potentials of B16 murine melanoma cell lines (42). ATF3 was recently shown to upregulate Twist/Snail/Slug expression and promote TGF-β-induced epithelial-to-mesenchymal transition in MCF10CA1a breast cancer cells (14) – a finding consistent with the role of ATF3 in promoting cancer progression. However, emerging evidence supports that ATF3 can also serve as a metastasis suppressor. Whereas we and others demonstrated that ATF3 represses pro-invasive matrix metalloproteinase-2 expression (21,43,44), ATF3 was shown to suppress migration, invasion and hepatic metastasis in colon cancer (19,45). A recent report demonstrated that ATF3 suppresses invasion of lung cancer cells and that ATF3 expression correlates with better survival of lung cancer patients (20). More recently, ATF3 was

found to suppress invasion and metastasis in bladder cancer by regulating cytoskeleton remodeling (18). These findings are in line with our results, supporting the metastasis-suppressing role of ATF3. Therefore, it appears that ATF3 contributes to cancer progression in a context-dependent manner. Such context-dependent regulation of cancer progression is not without precedent. Zbtb7a, a transcription factor previously shown to promote cellular transformation (46), was recently found to suppress invasion in prostate cancer (47). Interestingly, the bladder cancer cell lines used in the Yuan study (18) carry TP53 mutations. Whether the effects of ATF3 on cancer progression are dependent on TP53 mutation status is an interesting question and remains to be answered.

Acknowledgements – We would like to thank Drs. Bert Vogelstein and Dr. Shi-Yong Sun for providing materials. We also thank Dr. Gang Liu for technical guidance in microscopy. FOOTNOTES This work was supported in part by the NIH/NCI grants RO1CA139107 and RO1CA164006 to CY. HW was supported by the National Natural Science Foundation of China (grant No. 81202038) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars. The abbreviations used are: ATF, activating transcription factor; ATF3, activating transcription factor 3; co-IP, co-immunoprecipitation; CREB, cAMP responsive element binding protein; DBD, DNA-binding domain; GST, glutathione-S-transferase; HPV, human papillomavirus; IHC, immunohistochemistry; IP, immunoprecipitation; mutp53: mutant p53 protein; QPCR, quantitative polymerase chain reaction; shLuc, shRNA targeting luciferase; shp53, shRNA targeting p53; shRNA, short-hairpin RNA; TGFβ, transforming growth factor β; ZIP, leucine zipper domain REFERENCES

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Figure Legends Fig.1 ATF3 interacts with mutp53. A, schematic representation of the regions responsible for ATF3 binding to p53. B, Indicated GST-mutp53 fusion proteins were immobilized on glutathione agarose and incubated with in vitro-translated ATF3 for GST-pulldown assays. The lower panel shows Ponceau S staining. C, H1299 cells were transfected with ATF3 and indicated mutp53, and lysed for co-IP assays using the ATF3 antibody or IgG. Precipitated mutp53 proteins were detected with the DO-1 antibody. D, SK-BR-3 and A431 cells were treated with 1.5 µM of camptothecin for 4 h to increase the ATF3 expression level, and then subjected to co-IP assays using the ATF3 antibody or the p53 antibody as indicated. Fig.2 ATF3 downregulates NFKB2 expression and reverse drug resistance conferred by mutp53. A and B, Indicated cells were subjected to immunoblotting for p53 and ATF3 expression. C, Indicated H1299 cell lines were counted on various days to determine cell growth rates. D, Total RNAs were extracted, reverse-transcribed, and subjected to real-time PCR assays for NFKB2 expression. E, Cells were transfected with pRL-TK and a NF-κB luciferase reporter for dual luciferase activity assays. F, R175H-expressing (R175H) and control cells (V) were transfected with pRL-TK, the NF-κB luciferase reporter, and ATF3 as indicated for dual luciferase activity assays to determine NF-κB activity. G and H, Indicated cells plated in triplicate dishes were treated with DMSO, cisplatin (1 day), or etoposide (2 days), and then cultured for 9 days. Cell colonies were stained with crystal violet (G) and counted (H). I, Indicated cells treated with cisplatin or etoposide for 1 day were lysed for immunoblotting. J, R175H cells infected with Lentiviruses expressing shLuc or shATF3 (left), or SKBR3 cells transfected with siLuc or siATF3 (right), were treated with DMSO or etoposide for colony formation assays. The colony numbers of etoposide-treated cells were normalized to that of DMSO-treated cells. Immunoblots examining ATF3 and p53 levels in infected cells are also shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t test. Fig.3 ATF3 suppresses mutp53-induced cell migration and invasion. A, Migration of indicated cells into scratch wounds were recorded with a time-lapse video microscope. The movement of individual cells was presented as track-plots using ImageJ. B and C, The migration speed (B) and net distance (C) of individual cells were extracted from the track-plots using ImageJ. ns, no significant difference; **, p < 0.01; ***, p < 0.001. One-way ANOVA-Bonferroni test. D and E, Indicated cells were plated in Transwells for migration assays. Three independent experiments were carried out and representative fields are shown. The results were presented as mean ±SEM. *, p < 0.05, paired Student t test. F,

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Indicated cells were plated in Transwells coated with Matrigel for invasion assays. *, p < 0.05, Student t test. Fig.4 ATF3 suppresses migration of TP53-mutated cancer cells. A and B, A431 cells stably expressing shluc or shp53 were infected with retroviruses carrying pBabe or pBabe-ATF3, and subjected to immunoblotting (A) or scratch wound migration assays (B) as described in Fig 3A and 3B. C and D, A431 cells stably expressing shluc or shATF3 were subjected to immunoblotting (C) or scratch wound migration assays (D). E and F, MDA-MB-231 cells expressing shluc or shp53 were infected with pBabe or pBabe-ATF3 retrovirueses and subjected to immunoblotting (E) or migrating assays (F). ns, no significant difference; **, p < 0.01; ***, p < 0.001. ANOVA-Bonferroni test. Fig.5 ATF3 disrupts the mutp53-p63 interaction and reactivates p63 in mutp53 cells. A, R175H and R273H cells were transfected with FLAG-p63 and ATF3 as indicated, and subjected to co-IP assays using anti-FLAG M2 Affinity Gel. Amounts of p53 precipitated were quantitated by densitometry, normalized to precipitated p63 amounts, and presented in the right plot. B, H1299 cells were transfected with ATF3 and p63 as indicated, and subjected to co-IP assays using the ATF3 antibodyl. C, GST-p63 or GST immobilized on glutathione agarose was incubated with purified recombinant ATF3 for GST-pulldown assays. D, Indicated cells were transfected with p63, ATF3, a p63-responsive luciferase reporter (p53-luc), or pRL-TK as indicated for dual luciferase activity assays. F, Indicated cells were used to extract RNA for reverse transcription. cDNA was subjected to real-time PCR assays to determine relative SHARP1 mRNA level. ns, no significant difference; **, p < 0.01; ***, p < 0.001. Student t test. Fig.6 An ATF3 mutant deficient in mutp53 binding fails to suppress mutp53 function. A, Indicated GST-mutp53 fusion proteins were incubated with in vitro-translated ATF3 or ∆ATF3 for GST-pulldown assays. B, Immobilized GST-p63 or GST was incubated with purified recombinant ∆ATF3 for GST-pulldown assay. C, R175H or control cells were transfected with ATF3-IRES-GFP or ∆ATF3-IRES-GFP. Expression of GFP and ATF3 were determined by immunoblotting and fluorescence microscopy. D and E, R175H-expressing cells (R175H) or control cells (V) transfected with ATF3-IRES-GFP or ∆ATF3-IRES-GFP were subjected to scratch wound migration assays as described in Fig 3A, except that a fluorescence microscope was used to record cell movements. GFP-positive (ATF3+) and –negative (ATF3-) cells were tracked and used to calculate migration speed (C) and net distance (D). ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ANOVA-Bonferroni test. F, R175H cells transiently transfected with GFP, ATF3-IRES-GFP, or ∆ATF3-IRES-GFP were treated with DMSO or etoposide, and subjected to colony formation assays as described in Fig 2J. ns, no significant difference; *, p < 0.05. Student t test.. G, R175H cells was transfected with FLAG-p63, ATF3 or ∆ATF3 as indicated, and subjected to co-IP assays using anti-FLAG M2 Affinity Gel. H, Indicated cells were transfected with p63, ATF3, a p63-responsive luciferase reporter or pRL-TK as indicated for dual luciferase activity assays. Lower panel shows immunoblotting results of cell lysates. ***, p < 0.001. Student t test. I, R175 or control cells were transfected with GFP, ATF3-IRES-GFP, or ∆ATF3-IRES-GFP for 2 days. GFP-positive cells were sorted, and used for RNA preparation and QPCR assays to determine SHARP1 expression. ns, no significant difference; ***, p < 0.001. Student t test. Fig.7 ATF3 expression negatively correlates with metastasis in TP53 mutated lung cancer. A, Representative p53 staining in lung tumor samples. B, Representative low/no or high ATF3 staining in p53-postive tumor samples (#6193 and #2523). C, Summary of the staining results. The p values were calculated with the Fisher’s Exact test. D, Scoring of ATF3 staining based on intensity/nuclear localization in representative lung tumor sections. E, ATF3 staining scores were used to analyze the correlation between ATF3 expression and metastasis. ns, no significant difference. The p value was calculated with the Mann-Whitney test.

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V-ATF3

R17

5H-p

Babe

R17

5H-A

TF30

100

200

300

400

Dis

tanc

e( m

m)

*****

ns

V-pBab

e

V-ATF3

R27

3H-p

Babe

R27

3H-A

TF30

100

200

300

400***

***

ns

V-pBabe V-ATF3 R175H-pBabe R175H-ATF3 R273H-pBabe R273H-ATF3A

B C

V-pBabe V-ATF3

R175H-pBabe R175H-ATF3

V-pBabe V-ATF3

R273H-pBabe R273H-ATF3

D

E

V-pBabe V-ATF3

R175H-pBabe R175H-ATF3

F

Fig 3

V-pBab

e

V-ATF3

R17

5H-p

Babe

R17

5H-A

TF3

0

1

2

3

Re

lativ

eM

igra

tion

*

V-pBab

e

V-ATF3

R273H

-pBab

e

R273H

-ATF3

0

1

2

3

4

Rel

ativ

eM

igra

tion

*

V-pBab

e

V-ATF3

R17

5H-p

Babe

R17

5H-A

TF3

100

200

300

400

500

Ce

llN

um

be

r/fie

ld

*

16

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p53

ATF3

b-actin

pBab

e

ATF3

ATF3

pBab

eshLuc shp53

Fig 4

*****

pBab

e

ATF3

shp5

3-pB

abe

shp5

3-ATF3

0.0

0.2

0.4

0.6

0.8

1.0

Speed

( mm

/min

)

***

A

B

pBab

e

ATF3

ATF3

pBab

e

shLuc shp53

p53

ATF3

b-actin

E

1 2 3 4 1 2 3 4

pBab

e

ATF3

shp5

3-pB

abe

shp5

3-ATF3

0.00

0.05

0.10

0.15

0.20

Sp

ee

d(m

m/m

in)

**

ns

***

F

17

shluc

shATF3

0.0

0.2

0.4

0.6

0.8

Speed

( mm

/min

)

C

D***

p53

ATF3

b-actin

shLu

csh

ATF3

1 2

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p53

p63

ATF3

p53

p63

ATF3

IP: a-FLAG

Input

ATF3

FLAG-p63 - +- - +-- + + - + +

R175H R273H

R175H{ R273H{

w/o

ATF3

ATF3

w/o

ATF3

ATF3

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rela

tive

co-IP

ed

p53

Am

ount

1 2 3 4 5 6

Fig 5

A

D E***

**

V

R17

5H

R17

5H+A

TF3

V+ATF3

0.0

0.5

1.0

1.5

2.0

2.5

Rela

tive

Luc

ifera

seA

ctiv

ity

V

R27

3H

R27

3H+A

TF30.0

0.5

1.0

1.5 *****

V-pBab

e

V-ATF3

R17

5H-p

Babe

R17

5H-A

TF30.25

0.50

0.75

1.00

1.25

Rela

tive

mR

NA

leve

l ******ns

V-pBab

e

V-ATF3

R27

3H-p

Babe

R27

3H-A

TF30.25

0.50

0.75

1.00

1.25

Rela

tive

mR

NA

leve

l ****ns

B

Inpu

t

GST

GST-

p63

ATF3

p63

IgG

a-A

TF3

IgG

a-A

TF3

p63+ATF3

Inpu

t

p63

ATF3

1 2 3 4 5

C

1 2 3

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Inpu

tG

STR17

5H

R27

3H

WT

Inpu

t

GST

R17

5H

R27

3H

WT

ATF3

DATF3

1 2 3 4 5 6

p53

p63

ATF3

p53

p63

IP: a-FLAG

Input

ATF3

FLAG-p63 - +-- + +

R175H

1 2 3 4

DATF3 - +- --+

ATF3

DATF3

7 8 9 10

Fig 6

p53

p63

ATF3DATF3

ATF3DATF3

GFP

p53

ATF3-IRES-GFPDATF3-IRES-GFP - + - +

-+ -+

V R175H

V

R17

5H

R17

5H-A

TF3

ATF3

D

R17

5H+ V+A

TF3

ATF3

DV+

0.0

0.5

1.0

1.5

2.0

Re

lativ

eL

ucife

rase

Activ

ity

******

***

R17

5HAT

F3-

R17

5HAT

F3+

ATF3-

D

R17

5HAT

F3+

D

R17

5HV

ATF3-

VAT

F3+

ATF3-

DV

ATF3+

DV

0.0

0.1

0.2

0.3

0.4

Speed

( mm

/min

)

nsns

ns

*****

ATF3-IRES-GFP DATF3-IRES-GFP

A B

D

E

F

G

1 2 3 4 5 6

H

******

ns

V-GFP

R17

5H-G

FP

R17

5H-A

TF3

ATF3

D

R17

5H- V-A

TF3

ATF3

DV-

0.25

0.50

0.75

1.00

1.25

Rela

tive

Sha

rp1

mR

NA

leve

l

Inpu

t

GST

GST-

p63

DATF3

C

1 2 3

40

60

80

100

120

Co

lony

num

ber

GFP

ATF3-IR

ES-GFP

DATF3-

IRES-G

FP

*

ns

I

19

R17

5HAT

F3-

R17

5HAT

F3+

ATF3-

D

R17

5HAT

F3+

D

R17

5HV

ATF3-

VAT

F3+

ATF3-

DV

ATF3+

DV

0

100

200

300

Dis

tanc

e( m

m)

*ns

ns

*****

ns

***

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Fig 7

0

20

40

60

80

100Score=0

Score=1

Score=2

Score=3

AT

F3

Inte

nsit

y(%

per

gro

up

)

No Met Met No Met Met

p53-positive p53-negative

p =0.027 ns

A Ba-p53

p53

ATF3

#2523#6193

C

Score 0 Score 1

Score 2 Score 3

D

E

No metastasis Metastasis

(p-value)

- 2 9

+ 14 11 0.067

- 5 8

+ 11 12 0.73

ATF3 - 2 8

ATF3 + 9 4 0.036 *p53-negative

ATF3 - 0 1

ATF3 + 5 7 1.00

p53-positive

p53 staining

ATF3 staining

20

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Guohua Yu, Wei Wang, Dale D. Tang and Chunhong YanSaisai Wei, Hongbo Wang, Chunwan Lu, Sarah Malmut, Jianqiao Zhang, Shumei Ren,

Activating Transcription Factor 3 Suppresses the Oncogenic Function of Mutant p53

published online February 19, 2014J. Biol. Chem. 

  10.1074/jbc.M113.503755Access the most updated version of this article at doi:

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