The activating transcription factor 3 protein suppresses the oncogenic function of mutant p53 proteins

Abstract

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction 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 the 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 down-regulation 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.

Keywords: ATF3; Drug Resistance; Invasion; Lung Cancer; Migration; Mutant p53; p53.

FIGURE 1.

ATF3 interacts with mutp53. A, schematic representation of the regions responsible for ATF3 binding to p53. DBD, DNA-binding domain; ZIP, leucine zipper domain. B, the indicated GST-mutp53 fusion proteins were immobilized on glutathione-agarose and incubated with in vitro-translated ATF3 for GST pulldown assays. The bottom 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.

FIGURE 2.

ATF3 down-regulates NFKB2 expression and reverse drug resistance conferred by mutp53. A and B, the indicated cells were subjected to immunoblotting for p53 and ATF3 expression. V, control cells. C, the 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 an NF-κB luciferase reporter for Dual-Luciferase activity assays. F, R175H-expressing (R175H) and control cells 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, the 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, the 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 panel) or SKBR3 cells transfected with siLuc or siATF3 (right panel) 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. Immunoblot analyses examining ATF3 and p53 levels in infected cells are also shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test.

FIGURE 3.

ATF3 suppresses mutp53-induced cell migration and invasion. A, migration of indicated cells into scratch wounds was recorded with a time-lapse video microscope. The movement of individual cells are presented as track plots using ImageJ. B and C, 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 analysis of variance and Bonferroni test. D and E, the indicated cells were plated in Transwells for migration assays. Three independent experiments were carried out, and representative fields are shown. The results are presented as mean ± S.E. *, p < 0.05; paired Student's t test. F, the indicated cells were plated in Transwells coated with Matrigel for invasion assays. *, p < 0.05; Student's t test.

FIGURE 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. 3, A and B. 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 retroviruses and subjected to immunoblotting (E) or migrating assays (F). ns, no significant difference; **, p < 0.01; ***, p < 0.001; analysis of variance and Bonferroni test.

FIGURE 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. The precipitated amounts of p53 were quantitated by densitometry and normalized to precipitated p63 amounts and are presented in the right panel. B, H1299 cells were transfected with ATF3 and p63 as indicated and subjected to co-IP assays using the ATF3 antibody. C, GST-p63 or GST immobilized on glutathione-agarose was incubated with purified recombinant ATF3 for GST pulldown assays. D, the indicated cells were transfected with p63, ATF3, a p63-responsive luciferase reporter (p53-luc), or pRL-TK, as indicated, for Dual-Luciferase activity assays. E, the indicated cells were used to extract RNA for reverse transcription. cDNA was subjected to real-time PCR assays to determine the relative SHARP1 mRNA level. ns, no significant difference; **, p < 0.01; ***, p < 0.001; Student's t test.

FIGURE 6.

An ATF3 mutant deficient in mutp53 binding fails to suppress mutp53 function. A, the indicated GST-mutp53 fusion proteins were incubated with in vitro-translated ATF3 or ΔATF3 for GST pulldown assays. B, immobilized GST-p63 or GST was incubated with purified recombinant ΔATF3 for a GST pulldown assay. C, R175H or control cells (V) were transfected with ATF3-IRES-GFP or ΔATF3-IRES-GFP. Expression of GFP and ATF3 was determined by immunoblotting and fluorescence microscopy. D and E, R175H-expressing cells (R175H) or control cells transfected with ATF3-IRES-GFP or ΔATF3-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 GFP-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; analysis of variance and Bonferroni test. F, R175H cells transiently transfected with GFP, ATF3-IRES-GFP, or ΔATF3-IRES-GFP were treated with DMSO or etoposide and subjected to colony formation assays as described in Fig. 2J. *, p < 0.05; Student's t test. G, R175H cells were transfected with FLAG-p63, ATF3, or ΔATF3 as indicated and subjected to co-IP assays using anti-FLAG M2 affinity gel. H, the indicated cells were transfected with p63, ATF3, a p63-responsive luciferase reporter, or pRL-TK, as indicated, for Dual-Luciferase activity assays. Bottom panel, immunoblotting results of cell lysates. ***, p < 0.001; Student's t test. I, R175 or control cells were transfected with GFP, ATF3-IRES-GFP, or ΔATF3-IRES-GFP for 2 days. GFP-positive cells were sorted and used for RNA preparation and quantitative PCR assays to determine SHARP1 expression. ***, p < 0.001; Student's t test.

FIGURE 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-positive tumor samples (#6193 and #2523). C, summary of the staining results. The p values were calculated with Fisher's exact test. D, scoring of ATF3 staining on the basis of intensity/nuclear localization in representative lung tumor sections. E, ATF3 staining scores were used to analyze the correlation between ATF3 expression and metastasis (Met). ns, no significant difference. The p value was calculated with a Mann-Whitney test.