Ninjurin1, a Target of P53, Regulates P53 Expression and P53-Dependent Cell Survival, Senescence, and Radiation-Induced Mortality

Ninjurin1, a target of p53, regulates p53 expression and p53-dependent cell survival, senescence, and radiation-induced mortality

Seong-Jun Cho, Andrea Rossi, Yong-Sam Jung, Wensheng Yan, Gang Liu1, Jin Zhang, Min Zhang, and Xinbin Chen2

Comparative Oncology Laboratory, University of California, Davis, CA 95616

Edited by Carol Prives, Columbia University, New York, NY, and approved April 23, 2013 (received for review December 6, 2012) The tumor suppressor protein p53 plays a crucial role in coordi- in keratinocytes and dermal fibroblasts (16). These reports let us nating cellular processes, such as cell cycle arrest, apoptosis, and postulate that Ninj1 plays a role in cell survival and cell death. senescence. The nerve injury-induced protein 1 (Ninjurin1, Ninj1) However, whether p53 directly regulates Ninj1 expression and is a homophilic adhesion molecule and involved in nerve regen- whether Ninj1 has a role in the p53 pathway are not clear. eration. Interestingly, Ninj1 is found to be overexpressed in hu- In this study, we found that Ninj1 is a target of p53 and can be man cancer, but its role in tumorigenesis is not clear. Here, we induced by DNA damage in a p53-dependent manner. Most im- found that Ninj1 is transcriptionally regulated by p53 and can be portantly, silencing of Ninj1 led to increased p53 mRNA trans- induced by DNA damage in a p53-dependent manner. We also lation, resulting in p53-dependent cellular senescence, apoptosis, found that knockout or knockdown of Ninj1 increases p53 expres- and growth suppression. Moreover, mice heterozygous in ninj1 sion potentially through enhanced p53 mRNA translation. In addi- showed an increased radiosensitivity along with increased expres- tion, we found that Ninj1 deficiency suppresses cell proliferation sion of p53 in thymus upon whole-body γ-irradiation. Our findings but enhances apoptosis and premature senescence in a p53-depen- support the idea that Ninj1 plays a role in p53-mediated tumor dent manner. Consistent with this, we found that mice hetero- suppression in addition to nerve regeneration. zygous in ninj1 are hypersensitive to ionizing radiation-induced

lethality, along with increased expression of p53 in thymus. Results CELL BIOLOGY Taken together, we provided evidence that Ninj1 is a p53 target Ninj1 Is a p53 Target. To identify target genes regulated by p53, we and modulates p53 mRNA translation and p53-dependent pre- performed a microarray assay using H1299 cells that can inducibly mature senescence, cell proliferation, apoptosis, and radiation- express p53. Many well-defined p53 target genes were identified, induced mortality in vitro and in vivo. Thus, we postulate that as including p21, mouse double minute protein 2 (Mdm2), and a membrane adhesion molecule, Ninj1 is an ideal target to reg- Gadd45. In addition, we found that Ninj1 was induced by p53, ulate p53 activity via the p53-Ninj1 loop. consistent with a previous microarray study (10). To confirm the microarray study, H1299 cells were uninduced or induced to ex- cellular senescence | radiosensitivity press p53, followed by Northern blot analysis. We found that the level of Ninj1 transcript was markedly increased by p53, along with he tumor suppressor protein p53 plays a pivotal role in increased expression of p21 (Fig. 1A). Moreover, we found that in Ttumor suppression by regulating cell cycle arrest, apoptosis, response to treatment with camptothecin, the level of Ninj1 + − and senescence (1). Under unstressed condition, p53 protein transcript was increased in HCT116, p53 / HCT116, MCF7, and is maintained at a low level mostly through ubiquitination- LNCaP cells (Fig. 1B, compare lanes 3, 5, 11, and 13 with 4, 6, 12, mediated proteosomal degradation (2). In response to DNA and 14, respectively). These cells carry either one or two alleles of damage and other stress signals, p53 is activated and functions the wild-type p53 gene. As a positive control, p21 was also induced as a transcription factor to induce its downstream targets for various in these cells treated with camptothecin (Fig. 1B). By contrast, − − cellular processes such as cell cycle arrest [p21, growth arrest and Ninj1 was not induced in p53 / HCT116 and mutant p53-con- DNA-damage-inducible protein 45 (GADD45)] (3, 4), apoptosis taining T98G cells upon treatment with camptothecin (Fig. 1B, [p53 up-regulated modulator of apoptosis protein (PUMA), insulin- compare lanes 1 and 9 with 2 and 10, respectively). We also found − − like growth factor binding protein 3 (IGFBP3), DR5] (5–7), and that Ninj1 was induced in p21 / HCT116 cells treated with senescence [plasminogen activator inhibitor-1 (PAI-1), differentially camptothecin (Fig. 1B, compare lane 7 with 8), suggesting that the expressed in chondrocytes protein 1 (DEC1)] (8, 9). Although many induction of Ninj1 by p53 is not affected by loss of p21. Fur- p53 target genes related to various cellular processes have been thermore, quantitative RT-PCR showed that the level of Ninj1 identified, these are still insufficient to explain how p53 exerts its mRNA was increased by threefold in MCF7 cells upon treatment functions, in particular cellular senescence. Previously, many po- with doxorubicin, whereas p21 mRNA, as a positive control, was tential p53 target genes, including nerve injury-induced protein 1 increased by fourfold (Fig. 1C). Next, Western blot analysis was (Ninjurin1, Ninj1), were identified by a DNA microarray study (10). performed and showed that the level of Ninj1 protein was in- ninj1 The gene, which encodes a homophilic adhesion molecule creased in MCF7 cells by ectopically expressed p53 (Fig. 1D)and and cell surface protein, was found to be highly induced following nerve injury in dorsal root ganglion (DRG) neurons and Schwann

cells (11). Ninj1 protein is necessary for mediating homophilic Author contributions: S.-J.C., A.R., G.L., and X.C. designed research; S.-J.C., A.R., Y.-S.J., adhesion and promoting neurite outgrowth of DRG neurons (11, W.Y., G.L., and M.Z. performed research; S.-J.C., A.R., Y.-S.J., W.Y., J.Z., and X.C. analyzed 12), suggesting that Ninj1 plays a role in nerve regeneration. The data; and S.-J.C., J.Z., and X.C. wrote the paper. ninj1 gene was also found to be up-regulated in myeloid cells as- The authors declare no conflict of interest. sociated with experimental allergic encephalomyelitis and active This article is a PNAS Direct Submission. fi multiple sclerosis, which subsequently modulates the in ltration of 1Present address: Department of Medicine, School of Medicine, University of Alabama at inflammatory myeloid cells into central nerve system (13). Addi- Birmingham, Birmingham, AL 35295. tionally, Ninj1 was found to be overexpressed in hepatocellular 2To whom correspondence should be addressed. E-mail: [email protected]. carcinoma (14) and acute lymphoblastic B-cell leukemia (15). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Furthermore, Ninj1 was found to be induced by ionizing radiation 1073/pnas.1221242110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1221242110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Ninj1 is induced by p53. (A) Northern blots were prepared with total RNAs isolated from H1299 cells, which were uninduced (−)orinduced(+)to − − express p53 for 24 h. Northern blot analysis was performed with 32P-labeled cDNA probes derived from the Ninj1, p21,andGAPDH transcripts. (B) p53 / HCT116, p53+/−HCT116, p53+/+HCT116, p21−/−HCT116, T98G, MCF7, and LNCaP cells were untreated (−) or treated (+) with camptothecin (300 nM) for 12 h, followed by Northern blot analysis. (C) Quantitative RT-PCR was performed with total RNAs puriﬁed from MCF7 cells, which were untreated (Ctrl) or treated with doxorubicin (DOX) (200 ng/mL) for 12 h. The levels of Ninj1 and p21 transcripts were normalized to the GAPDH transcript level. The experiment was performed in triplicate. Error bars indicate SD. *P < 0.05 by two-tailed t test. (D) The levels of Ninj1, p53, Mdm2, and Actin proteins were measured by Western blots analysis in MCF7 cells uninduced (−)orinduced(+) to express p53 for 24 h. (E) MCF7 cells were untreated (Ctrl) or treated with doxorubicin (200 ng/mL) or camptothecin (200 nM) for 12 or 24 h and the levels of Ninj1, p53, Mdm2, and Actin proteins were measured by Western blot analysis. (F) HepG2 cells were treated with or without doxorubicin (200 ng/mL) for 12 or 24 h, and the levels of Ninj1, p53, and Actin proteins were measured by Western blot analysis. (G) MCF7 cells were treated with or without doxorubicin (200 ng/mL) for 12 h, and then cell membranes and nuclei were isolated. The levels of Ninj1 on the cell membrane (CM) and p53 in nucleus (NC) were measured by Western blot analysis. Claudin1 (Cldn1) and hnRNP c1/c2 proteins were measured as a cell membrane protein and a nuclear protein, respectively. (H) The subcellular localization of Ninj1 (green) was measured by immuno- ﬂuorescence staining in MCF7 cells treated without or with doxorubicin (200 ng/mL) or camptothecin (200 nM) for 12 h. The nuclei were visualized using 4′, 6-diamidino-2-phenylindole(DAPI).CPT,camptothecin.

upon treatment with doxorubicin and camptothecin (Fig. 1E, assay. We found that endogenous p53 directly bound to the Ninj1, compare lanes 1 and 4 with 2–3and5–6, respectively). Mdm2, as well as p21, promoters (Fig. 2B, lanes 4 and 6) but not to the awell-defined p53 target, was measured as a control and found to control GAPDH promoter (Fig. 2B, lane 3). Moreover, to test be increased (Fig. 1 D and E). Similarly, the level of Ninj1 protein whether the p53-RE in the Ninj1 promoter is responsive to p53, was increased in HepG2 cells, which carry wild-type p53, upon we generated two luciferase reporters carrying either wild-type or treatment with doxorubicin (Fig. 1F, compare lane 1 with 2 and mutant p53-RE under the control of the C-fos minimal promoter 3). Furthermore, we found that upon treatment with doxorubicin, (Fig. 2C). We showed that the luciferase activity under the con- the level of Ninj1 protein on the cell membrane was increased in trol of wild-type but not mutant p53-RE was markedly increased MCF7 cells (Fig. 1G) and HepG2 cells (Fig. S1). However, by wild-type p53, whereas mutant p53 was inert in both H1299 Claudin1, another cell adhesion molecule, was not increased by and MCF7 cells (Fig. 2D and Fig. S2). As a positive control, the DNA damage (Fig. 1G and Fig. S1). Similarly, we showed that luciferase activity under the control of the p21 promoter was in- Ninj1 protein was found to be localized on the MCF7 cell mem- creased by wild-type but not mutant p53 (Fig. 2D and Fig. S2). brane, and the intensity of Ninj1 staining was markedly increased Our findings suggest that p53 transcriptionally regulates the Ninj1 upon treatment with doxorubicin and camptothecin (Fig. 1H). gene by binding to the p53-RE in the promoter. p53 induces target gene expression via binding to specific DNA sequences in the promoter or intron. Thus, we searched Lack of Ninj1 Up-Regulates p53 Expression Potentially Through for p53-responsive element (p53-RE) in the genomic locus of Enhanced mRNA Translation. To determine the biological func- the Ninj1 gene and found one potential binding site at nucleotides tion of Ninj1, we generated stable cell lines in which shRNA −2573 to −2407 in the Ninj1 promoter (Fig. 2A). To determine against Ninj1 can be inducibly expressed in RKO cells as de- whether the Ninj1 promoter is recognized by p53, MCF7 cells scribed previously (17) and two clones were chosen for further were mock-treated or treated with camptothecin to induce p53 analysis. We showed that upon induction of Ninj1 shRNA, the expression, followed by chromatin immunoprecipitation (ChIP) level of Ninj1 transcript was decreased in RKO cells (Fig. S3A).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221242110 Cho et al. Downloaded by guest on September 28, 2021 + − contains most of the coding region (Fig. S7A). ninj1 / and − − ninj1 / mice were identified by genotyping using PCR (Fig. S7 B and C). Primary mouse embryonic fibroblasts (MEFs) were iso- + + + − − − lated from ninj1 / , ninj1 / ,andninj1 / embryos. We would like to mention that we were unable to detect mouse Ninj1 protein because of antibody limitation. Thus, the genetic status of the + + − − ninj1 gene in ninj1 / and ninj1 / MEFs at passage 4 (Fig. 3A) + + + − − − and in ninj1 / , ninj1 / ,andninj1 / MEFs at passage 3 (Fig. 3C) was confirmed by RT-PCR. Importantly, we found that the level − − of p53 was increased in ninj1 / MEFs at passage 4 compared with + + that in ninj1 / MEFs (Fig. 3B). Similarly, upon treatment with − − doxorubicin, the level of p53 was increased in ninj1 / MEFs at + − passage 3 and to a lesser extent in ninj1 / MEFs, compared with + + that in ninj1 / MEFs (Fig. 3D, compare lanes 1 and 4 with lanes 2–3and5–6, respectively). Consistently, the levels of p53 protein at both the basal and DNA-damage conditions were markedly − − increased in immortalized ninj1 / MEFs at passage 11, compared + + with that in ninj1 / MEFs (Fig. S8A, compare lanes 1 and 3 with 2 and 4, respectively). To confirm that Ninj1 modulates p53 expression, ninj1 was transiently knocked down in primary MEFs at passages 10 and 13 with two sets of siRNAs against ninj1 along with a scramble siRNA (Fig. 3E and Fig. S8B). Similarly, upon knockdown of ninj1, the level of p53 protein was markedly increased regardless of the passage of MEFs (Fig. 3F and Fig. S8C, compare lanes 1 and 4 with lanes 2–3and5–6, respectively). To explore the underlying mechanism by which Ninj1 regulates p53 expression, RT-PCR was performed and showed

p53 CELL BIOLOGY Fig. 2. p53 induces Ninj1 expression through binding to the p53-RE in the that the level of transcript was not altered in MEFs upon Ninj1 promoter. (A) Schematic presentation of the Ninj1 and p21 promoters knockout of Ninj1 and in RKO cells upon knockdown of Ninj1 along with the locations of the p53-RE and the primers used for ChIP assay. (Fig. S9 A and B). Moreover, we found that Ninj1 knockdown (B) ChIP assay was performed with MCF7 cells untreated (−) or treated (+) did not increase, but rather decreased, the half-life of p53 pro- with camptothecin (200 nM) for 12 h. The p53/DNA complexes were cap- tein in RKO cells (Fig. S9 C and D). These data let us postulate tured with anti-p53 (DO-1) and the binding of p53 to the p53-REs in the that Ninj1 regulates p53 mRNA translation. To test this, the ﬁ + + − − Ninj1 or p21 promoter was quanti ed by PCR. (C) Schematic presentation of level of newly synthesized p53 protein in ninj1 / and ninj1 / luciferase constructs carrying wild-type (Ninj1-W) or mutant p53-RE (Ninj1-M) MEFs was measured by 35S-metabolic labeling. Indeed, we found from the Ninj1 promoter. (D) The luciferase activity was measured from the luciferase constructs Ninj1-W or Ninj1-M in H1299 cells, which were that loss of Ninj1 led to 1.7-fold increase in the level of newly transfected with an empty vector or pcDNA3 expressing wild-type p53 or synthesized p53 in MEFs at passage 4 and 1.8-fold at passage 11 mutant p53(R249S) for 24 h. The luciferase construct carrying the p21 pro- (Fig. 3G and Fig. S9E). Similarly, upon knockdown of Ninj1 in moter was used as positive control. The experiment was performed in trip- RKO cells, the level of newly synthesized p53 protein was in- licate. Error bars indicate SD. *P < 0.001 between pcDNA3 and p53; **P < creased by 2.2-fold (Fig. 3H). Together, these data suggest that 0.001 between p53 and p53(R249S) by two-tailed t test. Ninj1 deﬁciency promotes p53 mRNA translation.

Ninj1 Deficiency Enhances Cellular Senescence and Radiosensitivity in Next, colony-formation assay was performed and showed that a p53-Dependent Manner. p53 is known to play a critical role in Ninj1 knockdown suppressed cell proliferation in RKO cells cellular senescence (1). Thus, to explore the biological signifi- regardless of treatment with doxorubicin (Fig. S3B). Because cance of Ninj1-mediated p53 expression, we measured cellular + + − − p53 is a potent growth suppressor, we reasoned that p53 may be senescence in primary ninj1 / and ninj1 / MEFs at various involved in growth suppression induced by Ninj1 knockdown. passages. We found that from passage 5 to 7, lack of Ninj1 To address this, the level of p53 protein was measured in RKO markedly increased the number of senescence-associated β- cells uninduced or induced to knock down Ninj1, along with galactosidase (SA-β-gal)–positive cells under a nonstress condi- treatment of doxorubicin or etoposide for various times. We tion (Fig. 4A, Upper). Quantitative analysis indicated that the + + found that the levels of p53 protein were markedly increased by SA-β-gal–positive cells were increased from 13.9% in ninj1 / − − Ninj1 knockdown upon DNA damage, concomitantly with in- MEFs to 36.2% in ninj1 / MEFs at passage 5, from 21.7 to creased expression of Mdm2 and p21 (Fig. S4 A–C). However, 40.3% at passage 6, and from 32.7 to 62.72% at passage 7 (Fig. knockdown of Ninj1 had little, if any, effect on the expression of 4A, Lower). We also found that lack of Ninj1 sensitized MEFs at Integrin β4 and β-catenin (Fig. S4D). Conversely, ectopic ex- passage 3 to DNA damage-induced premature senescence (50% in + + − − pression of Ninj1 decreased the levels of p53 protein along with ninj1 / MEFs vs. 82% in ninj1 / MEFs) (Fig. 4B). Consistent decreased expression of Mdm2 in RKO (Fig. S5). with this, we showed that the level of p53 protein along with p21, Because p53 is an apoptosis inducer, we postulate that Ninj1 PAI-1, and p130 was significantly increased by lack of Ninj1 in may mediate p53-dependent apoptosis. To test this, we measured MEFs at passage 4 at both the basal and stress conditions (Fig. 4C, the protein level of PUMA, a p53 target and a potent apoptosis compare lanes 1 and 3 with 2 and 4, respectively). We would like inducer, in RKO cells uninduced or induced to knock down Ninj1. to mention that p21, PAI-1, and p130 are well-defined markers for We found that Ninj1 knockdown increased the levels of p53 and p53-dependent cellular senescence (8, 18). PUMA proteins upon treatment with doxorubicin (Fig. S6A) and, Because p53 is known to be critical for radiosensitivity (19), we consequently, enhanced DNA damage-induced apoptosis (5.5% explored whether loss of Ninj1 predisposes mice to radiation- vs. 15.0%) (Fig. S6B). induced lethality upon whole-body γ-irradiation. We would like To verify the regulation of p53 by Ninj1, we generated ninj1- to mention that Ninj1-null mice were short-lived, potentially + − deficient mice by deleting exon 2 in the ninj1 gene, which because of hydrocephalus (Fig. S10). Thus, ninj1 / mice (n =

Cho et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 11), which did not exhibit any detectable abnormality by 1 y of + + age, along with ninj1 / mice (n = 9), were irradiated with 8 Gy of whole-body γ-ray and then monitored for radiation-associated + − mortality for 22 d. We found that ninj1 / mice were more sen- + + sitive to radiation-induced death than ninj1 / mice (Fig. 4D). + − The median survival time was 13 d for ninj1 / mice vs. 19 d for + + + − ninj1 / mice. The difference in median survival between ninj1 / + + and ninj1 / mice was statistically significant (log-rank test, P = 0.043). Consistent with this, we found that the level of p53 protein + − + + in the thymus was higher in ninj1 / mice than in ninj1 / mice upon exposure to 8 Gy of γ-ray (Fig. 4E, compare lanes 1 and 3 with 2 and 4, respectively). In addition, we found that the level of ninj1 mRNA in thymus was increased by γ-irradiation compared with unirradiated control thymus (Fig. S11), consistent with the observation that ninj1 can be induced by DNA damage (Fig. 1B). These data suggest that Ninj1 deficiency enhances ionizing radiation-induced p53 expression and lethality in mice. To further explore the involvement of p53 in Ninj1-mediated biological function, we determined whether cellular senescence induced by loss of Ninj1 (Fig. 4 A–C) requires p53. To test this, we generated Ninj1 and p53 double-knockout MEFs. We showed − − that ninj1 transcript and p53 protein were absent in ninj1 / ; − − p53 / MEFs regardless of doxorubicin treatment (Fig. 5 A and B, lanes 3 and 6). We also showed that loss of Ninj1 increased the − − number of SA-β-gal–positive cells in ninj1 / MEFs at passage 5 + + compared with that in ninj1 / MEFs (Fig. 5 C and D), consistent with the observation in Fig. 4A. Most importantly, cellular senescence induced by loss of Ninj1 was abrogated by p53 knockout regardless of treatment with doxorubicin (Fig. 5 C and D). Con- sistent with this, the increased levels of senescence markers p130 and p21 by loss of Ninj1 were mitigated by p53 knockout regardless of treatment with doxorubicin (Fig. 5B, compare lanes 2 and 5 with 3 and 6, respectively). These data suggest that Ninj1 regulates cellular senescence in a p53-dependent manner. Discussion p53 plays a critical role in growth suppression, and disruption of balanced p53 regulation would lead to tumor development. Thus, the p53 activity needs to be finely tuned in normal tissues. To tightly control the p53 pathway with complex downstream networks, various positive or negative autoregulatory feedback loops are involved in the regulation of the p53 pathway (20), including p53-Mdm2 (21), p53-RNPC1 (22), p53-Wig-1 (23), and p53-Ninj1 in this study. Specifically, we found that p53 can transcriptionally up-regulate Ninj1 by directly binding to the p53- RE in the promoter. In addition, we showed that silencing of Ninj1 enhances p53 mRNA translation, leading to increased p53 expression. Furthermore, we showed that Ninj1 deficiency suppresses cell proliferation but enhances apoptosis and premature cellular senescence in a p53-dependent manner. Finally, we showed that mice deficient in Ninj1 are hypersensitive to ionizing radiation-induced lethality along with increased expression of p53 in thymus. Together, we uncovered a feedback loop between p53 and Ninj1, and our data suggest that Ninj1 plays a role in p53- dependent cell proliferation, premature senescence, and radio- Fig. 3. Lack of Ninj1 enhances p53 expression potentially through mRNA sensitivity in vitro and in vivo (Fig. 5E). + + − − translation. (A) Total RNA was isolated from ninj1 / and ninj1 / MEFs at passage 4 and the levels of ninj1 and Gapdh transcripts were measured by +/+ −/− RT-PCR. (B) The levels of p53 and Actin in ninj1 and ninj1 MEFs at of ninj1 and actin transcripts were measured by RT-PCR. The siRNA sequence passage 4 were measured by Western blot analysis. (C) Total RNA was iso- + + + − − − targeting two separate regions in the ninj1 gene is described in Table S2. lated from ninj1 / , ninj1 / , and ninj1 / MEFs at passage 3, and the levels of +/+ +/− (F) Experiment was performed as described in E, and the levels of p53 and ninj1 and Gapdh transcripts were measured by RT-PCR. (D) ninj1 , ninj1 , 35 −/− Actin were analyzed by Western blot analysis. (G) S metabolic-labeling + + − − and ninj1 MEFs at passage 3 were treated without (Ctrl) or with doxo- assay was performed with ninj1 / and ninj1 / MEFs at passage 4 as de- rubicin (DOX) (200 ng/mL) for 24 h, and the levels of p53 and Actin were scribed in Materials and Methods.Thelevelofnewlysynthesizedp53 analyzed by Western blot analysis. (E) Total RNA was isolated from MEFs protein was measured and quantified by densitometric analysis. NS indi- (passage 10) transiently transfected with scrambled siRNA (SCR) (50 nM) or cates nonspecificband.(H) The experiment was performed as in G except siRNA (50 nM) against ninj1 (siNinj1-1 or siNinj1-2) for 48 h, followed by that RKO cells uninduced (−) or induced (+) to express shRNA against Ninj1 treatment without (Ctrl) or with doxorubicin (200 ng/mL) for 18 h. The levels for 3 d were used.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221242110 Cho et al. Downloaded by guest on September 28, 2021 CELL BIOLOGY

+ + − − Fig. 4. Lack of Ninj1 promotes cellular senescence and radiosensitivity. (A, Upper) ninj1 / and ninj1 / MEFs at passage 5–7wereanalyzedbySA-β-gal assay. (A, Lower)Quantiﬁcation of SA-β-gal–positive cells as shown A, Upper. Error bars indicate SD. *P < 0.0001 by two-tailed t test. (B, Upper) ninj1+/+ and ninj1−/− MEFs at passage 3 were untreated (Ctrl) or treated with doxorubicin (DOX) (50 ng/mL) for 3 d, followed by SA-β-gal assay as described in Materials and Methods.(B, + + − − Lower)Quantiﬁcation of SA-β-gal–positive cells as shown in B, Upper. Error bars indicate SD. *P < 0.0001 by two-tailed t test. (C) ninj1 / and ninj1 / MEFs at passage 4 were untreated (Ctrl) or treated with doxorubicin (200 ng/mL) for 24 h, and the levels of p53, p21, p130, PAI-1, and Actin were measured by Western + + + − + + + − blot analysis. (D) Kaplan–Meier survival analysis of ninj1 / and ninj1 / mice after 8 Gy of whole-body γ-ray. P = 0.043 by log-rank test. (E) ninj1 / and ninj1 / mice were irradiated with 8 Gy and then killed 3 h later. Thymus was isolated, and the levels of p53 and Actin were measured by Western blot analysis.

Numerous observations suggest that cell–cell or cell–matrix modulate the assembly of translation initiation complex such as adhesion through adhesion molecules, such as integrins and eIF4F complex (4E+4G+4A), eIF4B, and poly(A)-binding cadherins, regulates cell survival, tumor invasion, and metas- protein (33). For example, RNPC1, also called Rbm38, is a p53 tasis(24,25).Interestingly,theabilityofp53toregulatecell target, which, in turn, represses p53 mRNA translation by pre- survival in response to DNA damage is also regulated by cell– venting eIF4E from binding to p53 mRNA (22). cell interaction via adhesion molecules (26). In addition, focal Ninj1 is known to be highly expressed in hepatocellular car- adhesion kinase, an integrin- and growth factor-associated tyro- cinoma (14) and acute lymphoblastic leukemia (15), suggesting sine kinase, is known to promote cell survival through enhancing that Ninj1 has an oncogenic potential. In this study, we showed p53 degradation (27). Although p53 regulates a diverse array of that lack of Ninj1 leads to increased expression of p53 and sub- target genes (28), including the gene encoding integrin α5 (29), it sequently enhances cellular senescence, growth suppression, and is still not clear whether p53 directly regulates the gene encoding radiation-induced lethality in mice. Considering that p53 plays a cadherin. Here, we showed that Ninj1, a cell adhesion molecule, a critical role in growth suppression and radiation-induced he- ﬁ is regulated by p53, which, in turn, regulates cell proliferation matopoietic injury (19, 34), our ndings indicate that Ninj1 exerts and premature senescence through modulating p53 expression. its oncogenic function by repressing p53-dependent growth sup- Nevertheless, the mechanism by which Ninj1 regulates p53 mRNA pression. Overall, we postulate that as a membrane adhesion translation is not clear but worth further investigation. One pos- molecule, Ninj1 is an ideal target to regulate p53 activity via the sibility is via mechanistic target of rapamycin (mTOR) kinase, p53-Ninj1 loop for cancer therapy. which can be activated by growth signals and regulates mRNA Materials and Methods translation via assembly of eukaryotic translation initiation Plasmid constructs, cell line generation with tetracycline-regulated inducible factor (eIF) complex (30). For example, inhibition of mTOR γ p53 system, ninj1 knockout mouse generation, MEF isolation, -irradiation, decreases translation (31) whereas knockout of protein Northern and Western blot analyses, ChIP assay, luciferase assay, RT-PCR, regulated in development and DNA damage response 1, a SA-β-gal assay, 35S metabolic-labeling assay, colony-formation assay, cell stress-induced inhibitor of mTOR1, increases p53 translation fractionation, immunoﬂuorescence analysis, DNA histogram analysis, and (32). The other possibility is that Ninj1-mediated signals may statistical analysis are available in SI Materials and Methods. Primers for

Cho et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 + + − − − − Fig. 5. Lack of Ninj1 enhances cellular senescence in a p53-dependent manner. (A) The levels of ninj1 and Gapdh mRNAs in ninj1 / , ninj1 / , and ninj1 / ; − − + + − − − − − − p53 / MEFs at passage 4 were measured by RT-PCR. (B) ninj1 / , ninj1 / , and ninj1 / ;p53 / MEFs at passage 5 were untreated (Ctrl) or treated with + + − − − − doxorubicin (DOX) (200 ng/mL) for 18 h, and the levels of p53, p21, p130, and Actin were measured by Western blot analysis. (C) ninj1 / , ninj1 / , and ninj1 / ; − − p53 / MEFs at passage 5 were untreated (Ctrl) or treated with doxorubicin (50 ng/mL) for 3 d, followed by SA-β-gal assay. (D) Quantiﬁcation of SA-β-gal– positive cells as shown in C. Error bars indicate SD. *P < 0.0001 between ninj1+/+ and ninj1−/−;**P < 0.0001 between ninj1−/− and ninj1−/−;p53−/− by two-tailed t test. (E) Model for Ninj1 to regulate p53-dependent cellular senescence, cell survival, and radiosensitivity.

RT-PCR and ninj1 genotyping are listed in Table S1.siRNAsequencesfor ACKNOWLEDGMENTS. This work was supported, in part, by National Institutes mouse ninj1 knockdown are listed in Table S2. of Health Grants CA076068 and CA102188.

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