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Neoplasia. Jan 2009; 11(1): 22–31.
PMCID: PMC2606115

Blocking of p53-Snail Binding, Promoted by Oncogenic K-Ras, Recovers p53 Expression and Function1,2

Abstract

Differentially from other kinds of Ras, oncogenic K-Ras, which is mutated approximately 30% of human cancer, does not induce apoptosis and senescence. Here, we provide the evidence that oncogenic K-Ras abrogates p53 function and expression through induction of Ataxia telangiectasia-mutated and Rad3-related mediated Snail stabilization. Snail directly binds to DNA binding domain of p53 and diminishes the tumor-suppressive function of p53. Thus, elimination of Snail through si-RNA can induce p53 in K-Ras-mutated cells, whereas Snail and mutant K-Ras can suppress p53 in regardless of K-Ras status. Chemicals, isolated from inhibitor screening of p53-Snail binding, can block the Snail-mediated p53 suppression and enhance the expression of p53 as well as the transcriptional activity of p53 in an oncogenic K-Ras-dependent manner. Among the chemicals, two are very similar in structure. These results can answer why K-Ras can coexist with wild type p53 and propose the Snail-p53 binding as the new therapeutic target for K-Ras-mutated cancers including pancreatic, lung, and colon cancers.

Introduction

In human cancer, the oncogenic mutation of Ras family genes including H-, N-, and K-Ras, is frequently detected [1]. In particular, K-Ras mutation is frequent event in pancreatic cancer (80–90%), lung adenocarcinoma (35%), and colon cancer (40%) [2–4]. However, in previous studies, it has been revealed that oncogenic Ras induces senescence and apoptosis through p53 activation [5,6]. Thus, without functional defect of p53, activated Ras cannot promote tumor formation. Indeed, hepatocellular carcinoma, induced by oncogenic H-Ras, is rapidly regressed by restoration of p53 [7,8]. However, p53 can coexist with oncogenic K-Ras in human cancer tissues and cell lines [2,9,10]. In a mouse model, the physiological level of oncogenic K-Ras can evoke adenoma despite the intact p53 system [11,12]. These results suggest that K-Ras may have a unique function differentially from other Ras (H- or N-Ras).

About the p53 regulation network, overexpression of Mouse double minute 2 (MDM2) is one of the well-confirmed mechanisms for p53 suppression [13]. Because p53 is rapidly degraded by MDM2 that promotes p53 ubiquitinylation and degradation, overexpression of MDM2 and p53 mutation shows a mutually exclusive pattern [14]. However, MDM2 overexpression, which is achieved through DNA amplification, is a rare event in human carcinoma (instead, MDM2 amplification is frequently detected in human and mouse sarcomas) [15,16]. Another p53 suppression mechanism of human cancer is silencing or deletion of p14/ARF, an inhibitor of MDM2 [17,18]. Thus, loss of p14/ARF results in p53 suppression through the hyperactivation of MDM2. Moreover, the induction of p19/ARF (homolog of human p14/ARF) has been suggested to be responsible for Ras-induced p53 activation [19,20]. However, p14/ARF cannot be induced by oncogenic Ras in human cells, and it shows the p53-independent methylation or deletion pattern in human cancer [21], suggesting that in the human system, p14/ARF function is not responsible for oncogene-induced p53 activation. Moreover, p14/ARF knock-out mouse shows quite a different cancer spectrum with the p53-deficient animal [22], also suggesting the independent role of both proteins.

As mentioned above, N- or H-Ras-mediated tumorigenesis is accompanied with loss of p53 function, whereas oncogenic K-Ras can coexist with wild type p53. The purpose of this study was to answer how it is possible. To do this, we assume that K-Ras might possess a unique function that can permit the cell to escape p53-mediated cellular senescence or apoptosis. In this study, we reveal that oncogenic K-Ras suppresses p53 through the induction of Snail. Although Snail is a transcriptional repressor [23], Snail, stabilized by K-Ras-Ataxia telangiectasia-mutated and Rad3-related (ATR) pathway, binds to and eliminates p53 in a transcription-independent mechanism. Furthermore, we identify the chemicals that can block the interaction between p53 and Snail and can induce p53 expression in K-Ras-mutated cell lines.

Materials and Methods

Isolation of Mouse Fibroblast and Immortalization

A 6-month-old male mouse was killed to collect fibroblast. After isolation of lung, tissue was chopped and dissociated using culture-mess. After 3 days of incubating in Dulbecco's modified Eagle's medium (DMEM) containing 20% FBS, attached cells were seeded in culture dishes and transfected with mutant H-, N-, and K-Ras using JetPEI (Polyplus Transfection, New York, NY) following the manufacturer's protocol. After 72 hours, we initiated selection using 400 µg/ml of G418 containing DMEM.

Cell Culture and Reagents

Cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained in RPMI-1640 or DMEM containing 10% FBS. Antibodies used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) or Cell Signaling (p53-R, p-Erk; Danvers, MA). Ras expression vectors and Snail vectors were provided by Dr. Chi SG (Korea University) [24] and Hung M-C (University of Texas) respectively [25]. p53 S46D and 46A were provided by Mayo LD (Case Western Reserve University) [26]. Chemicals used in this study were purchased from Calbiochem (San Diego, CA). Recombinant p53 was obtained from Assay Designs (Ann Arbor, MI).

Immunostaining and Western Blot Analysis

For cell staining, we routinely washed and fixed with 100% Me-OH and incubated with antibodies (first antibody (Ab): 1:200, overnight at 4°C; secondary Ab: 1:1000, 2 hours at room temperature). To detect secreted p53 and Snail, HCT116 p53-/- cells were transfected with vectors for 24 hours in 1 ml of RPMI 1640 and fixed by adding 1 ml of 2% paraformaldehyde (PFA) without washing. After fixation, cells were washed briefly with PBS twice and incubated with blocking buffer (PBS + anti-human Ab (1:500)) to eliminate non-specific binding. After washing with PBS, cells were incubated with anti-p53 and anti-Snail Ab and matched secondary Ab. For protein analysis, we extracted protein using a radioimmunoprecipitation assay (RIPA) buffer and applied the sample to SDS-PAGE, and followed the routine Western blot protocol. Immunoprecipitation analysis was performed using the general protocol. In brief, cell lysate was incubated first with Ab for 4 hours at 4°C and then with protein-A/G-agarose for 2 hours. After centrifugation and washing three times, the precipitated complex was subjected to SDS-PAGE/Western blot analysis.

Transfection and si-RNA

For cell transfection, we used jetPEI according to the manufacturer's protocol. Cells were incubated with DNA/jetPEI mixture for 24 hours in complete media. For in vitro gene knock out, we generated si-RNA against Snail [27] and MDM2 [28]. Using jetPEI, we transfected si-RNA and checked the effect after 24 hours.

Far Western Blot and In Vitro Kinase or Binding Assay

To address direct binding between Snail and p53, we prepared the membrane, which is loaded with recombinant p53 or Snail or p53-transfected cell lysate through typical SDS-PAGE and gel transfer method. After blocking with 5% nonfat dry milk, the membrane was incubated with p53 or Snail-transfected p53-/- HCT116 cell lysate for 4 hours at 4°C. After washing, the membrane was subjected to typical Western blot procedure with p53 Ab or Snail Ab. For in vitro binding, the recombinant p53 and GST-Snail were incubated for 1 hour at 4°C with rotation; we performed immunoprecipitation with p53 Ab or GST Ab and Western blot analysis with GST or p53 Ab. To examine the modification of Snail, 293 cells were used for transfection. After fraction or lysis, lysates were incubated with GST or GST-Snail for 1 hour at 25°C and were subjected into SDS-PAGE and Western blot analysis. Antibodies against p-mitogen-activated protein kinase (MAPK) and p-ataxia-telangiectasia mutated/ATR substrates were obtained from Cell Signaling.

Recombinant Proteins and GST Pull-Down

Three human Snail fragments (residues 1–90, 91–112, and 113–264) and p53 fragments (1–93 and 93-292) were expressed in Escherichia coli as a GST fusion protein. Each fragment was loaded on to GSH-agarose and then eluted using a buffer containing 20 mM reduced glutathione after extensive washing. The eluted fractions were further purified using an anion exchange chromatography (HitrapQ; GE Healthcare Biosciences, Piscataway, NJ). The recombinant human p53 protein (residues 94–292) was expressed in E. coli using the vector pET28A, which contains a hexa-histidine tag at the C-terminus. The p53 protein was purified using Ni-NTA affinity and a size exclusion chromatography (Superdex 200; GE Healthcare Biosciences, Piscataway, NJ). To address the direct binding between p53 and Snail, agarose bead-conjugated GST or GST-Snail was incubated with cell lysate or His-p53 in RIPA for 45 minutes at 4°C. After washing with PBS and RIPA, the precipitated protein was subjected to SDS-PAGE and Western blot.

Chemical Screening

To isolate Snail-p53 binding inhibitor, we generated ELISA system. We immobilized His-p53 (93–292) on a 96-well plate using 0.5% PFA. After drying and washing, we incubated with GSTSnail with 0.1 µM of chemicals (final concentration). After a 1-hour incubation, the 96-well plates were washed with TBS-T and were incubated with anti-GST-Ab (1:10,000 for 45 minutes) and anti-mouse-IgG-HRP (1:50,000 for 30 minutes). After washing twice, plates were incubated with 3,3′,5,5′ tetramethylbenzidine (TMB) solution and Stop solution. Using the ELISA reader, we determined the value.

Results

Oncogenic K-Ras Suppresses p53

To test our hypothesis, we tried to generate a stable cell line using normal mouse fibroblast. Transfection of N-Ras or H-Ras induced apoptosis or senescence (Figure W1A). This result is consistent with others' previous result that single oncogene or oncogenic Ras did not produce immortalized cell [29,30]. However, K-Ras12V-transfected cells were growing and were maintained until now (>6 months from transfection of K-Ras12V; Figure W1A). Because we used the normal adult lung fibroblast and selected the transfected cells using G418, proliferating cells did not result from spontaneous immortalized cell. To address how K-Ras-transfected cells overcame oncogene-induced senescence and apoptosis, we examined the effect of K-Ras on p53 function. Forced expression of oncogenic K-Ras suppressed the p53 expression in wild type p53-containg cell lines (Figure 1A). To confirm this, we measured the expression of exogenous p53 after cotransfection with oncogenic Ras. Different from H- or N-Ras, K-Ras could evoke p53 suppression, which was not blocked by si-MDM2 (Figure 1B). We could also obtain the similar result from p53-deficient HCT116 (data not shown). However, wild type K-Ras did not suppress p53 expression (Figure 1, C and D). Reduction of p53 occurred using K-Ras in a dose-dependent manner (Figure W1B). Blocking of Ras activity through dominant-negative Ras (DN-Ras) could increase the p53 expression only in K-Ras-mutated A549 but not in HepG2 (Figure 1E). Because A549 is a p14/p16-deficient cell line [31], induction of p53 by DN-Ras was not related with the p14-MDM2 pathway. In other kinds of wild type K-Ras cell lines (MCF-7 and MKN-45), we did not observe the induction of p53 by DN-Ras (data not shown). This result suggests that endogenous oncogenic K-ras suppresses p53 expression. However, DN-Ras did not show an obvious and synergic effect on DNA damage-mediated p53 suppression (Figure W1C), implying that a strong genotoxic stress could overcome the oncogenic K-Ras-mediated p53 suppression. K-Ras-mediated p53 suppression was detected in point mutant p53 (S46A, 22/23, and 175; Figure 1, F and G). However, p53 S46D, the active form of p53, showed resistance to K-Ras-mediated p53 suppression (Figure 1F). This result is consistent with our previous result that genotoxin-induced p53 activation could overcome K-Ras-mediated suppression (Figure W1C). Because the 22/23 mutant does not associate with MDM2 [32], we can confirm that K-ras-mediated p53 suppression is achieved through an MDM2-independent pathway. Proteasome inhibitors did not block the K-Ras-mediated p53, also indicating the irrelevance of MDM2 or p53 ubiquitin system (Figures 1H and W2A). We also checked the effect of MAPK signaling inhibitors on K-Ras-mediated p53 suppression. However, blocking of the MAPK pathway did not abolish the effect on K-Ras-mediated p53 suppression (Figure W2, B–E). These results implied that K-Ras-mediated p53 suppression would be achieved through a novel pathway, independently from canonical Ras-MAPK pathway or MDM2-mediated negative feedback loop.

Figure 1
Suppression of p53 by oncogenic K-Ras. (A) Oncogenic K-Ras suppresses endo-p53 in several kinds of cell lines. Cells were transfected with K-Ras 12V or EV vector for 24 hours. (B) K-Ras but not H- and N-Ras suppresses p53 expression. Suppressed p53 is ...

Snail Is Responsible for K-Ras-Mediated p53 Suppression

To explore the molecular mechanism of p53 reduction, we checked the interaction between p53 and K-Ras or localization of K-Ras. However, we did not observe the binding between p53 and K-Ras or nuclear translocation of oncogenic K-Ras (data now shown). Through searching for literatures, we found that Snail can negatively regulate p53 [27]. Because p53 should be inactivated during cancer progression [33], and Snail can promote epithelial-mesenchymal transition (EMT) that permits the cancer cell to migration and metastasis [23,34,35], we examined the relationship between p53 and Snail. Moreover, the expression of Snail is elevated in reoccurred cancer, where p53 should be inactivated [36]. This result encouraged us to investigate the relevance of p53 and Snail. First, we monitored the effect of K-Ras on Snail expression and revealed that Snail could be induced by K-Ras (Figure 2A). Next, we measured the effect of Snail on p53 expression in cell lines. Overexpression of Snail could suppress p53 in A549 and HepG2 cell lines, whereas Snail knock down induced p53 only in A549 (oncogenic K-Ras-containing cell line) but not HepG2 (Figure 2B). In addition, si-Snail could increase the sensitivity to DNA damage agent (Figure W2F). Thus, overexpression of Snail could promote cell proliferation and render the resistance to DNA damage-induced cell death (Figure W2G). Snail could also suppress the exo-p53 as well as endo-p53, similarly to K-Ras (Figure 2C). A more interesting feature was that Snail and p53 were reduced together when they were cotransfected (Figure 2, D and E) regardless of mutant p53 (Figure 2F). However, mRNA of Snail and p53 were not reduced (Figure 2, C and D).We also examined the effect of Snail on p53 transcript. However, Snail did not reduce p53 mRNA (Figure W2H). These results indicated that although p53 and Snail were well-confirmed transcriptional regulators, their reduction was irrelevant with transcriptional regulation. In addition, elimination of Snail could block the K-Ras-mediated p53 suppression (Figure 2, G and H). We could also obtain the similar result from exo-p53 (Figure W3, A and B). These results indicate that K-Ras-mediated p53 suppression is achieved trough Snail induction. To exclude the possibility of technical artifact of cotransfection, we examined the expression of both proteins from the early phase. Within 4 hours, oncogenic K-Ras could induce p53, whereas p53 was reduced after 6 hours (Figure W3C). This result indicated that p53 suppression was not achieved by transfection artifact but was an effect of transfected proteins. Reverse transcription-polymerase chain reaction analysis suggested that Snail or p53 was not regulated at the transcription level (Figure W3D). We also checked the apoptosis and cell cycle in K-Ras/Snail-transfected cells. However, apoptosis and cell cycle inhibition were not obviously induced by K-Ras/Snail (Figure W3E; see 4′-6-Diamidino-2-phenylindole staining in Figures 1D and and2E,2E, and propidium iodide in Figure 2H; data not shown). We could also observe the reduction of p53 by K-Ras/Snail in aphidicolin-treated cell (data not shown) suggesting that the reduction of p53 was not linked to cell cycle. Next, we monitored the effect of Snail on the half-life of p53 through cycloheximide pulse chase. However, Snail did not shorten p53 half-life (Figure W3F). From our previous result (Figure 1F), we showed the resistance of p53 S46D against K-Ras-mediated suppression. Thus, we checked the effect of Snail on the expression of p53 S46D and found out that Snail, differentially from K-Ras, could suppress p53 S46D expression (Figure W4A). To reveal the reason, we monitored the effect of p53 S46D on Snail expression and found that S46D could suppress Snail expression at the transcription and translation levels (Figure W4B). Thus, differentially from wild type p53, in which si-Snail could restore the p53 suppression, si-Snail did not induce p53 expression when S46D was transfected (Figure W4C). Our results indicate that under certain stress condition, activated p53 by modifying at the serine 46 residue can overcome K-Ras-mediated suppression mechanism by the repression of Snail transcript. This mechanism would be one of the protecting role of p53 against K-Ras-mediated tumorigenesis. However, until now, we did not know what kinds of cellular stresses can overcome K-Ras-Snail-mediated p53 suppression by activating p53 S46D. This result also provides the clue why, in a considerable portion of human cancers, p53 should be mutated or deleted during the transition from in situ carcinoma to metastatic advanced cancer despite oncogenic K-Ras harboring cancers [2].

Figure 2
Snail suppresses p53. (A) Forced expression of oncogenic K-Ras can induce Snail expression, similarly with ALLN treatment. HCT116 p53- cells were incubated with ALLN for 6 hours or transfected with the indicated vectors for 24 hours. (B) Snail can regulate ...

Induction of Snail Is Achieved through ATR

To address how K-ras induce Snail, we first examined the engagement of AKT, because Ras can activate AKT, which can suppress GSK-3β-mediated Snail destabilization [25]. However, AKT-KD did not block the Snail or K-Ras-induced p53 suppression (Figure 3A). In contrast, suppression of ATR through si-RNA could block the p53 suppression (Figure 3B). Indeed, Snail was increased by ATR but not by ataxia-telangiectasia mutated (ATM) and nocodazole treatment (Figure 3, C and D). In vitro kinase assay showed that Snail was phosphorylated by ATR (Figure W4D). K-Ras, which has been known to activate ATR [31], also increased p-Snail in an ATR-dependent manner (Figure 3E) and extended the half-life of Snail (Figure 3F). These results suggested that K-Ras regulated Snail through ATR.

Figure 3
Requirement of ATR activity in K-Ras-induced Snail stabilization. (A) KD-AKT does not block the K-Ras or Snail-mediated p53 suppression. However, si-Snail can block the K-Ras and Snail-mediated p53 suppression. (B) Si-ATR can block the K-Ras-mediated ...

Direct Interaction between Snail and p53

Our next question was how Snail suppressed p53. Because Snail is a nuclear protein, and Snail and p53 disappeared when they were cotransfected (Figure 2, D–F), we checked the interaction between them. From endo-IP, we found that these proteins could be associated (Figure 4A). FarWestern blot analysis and GST pull-down assay indicated that Snail and p53 directly interacted with each other (Figure 4, B and C). Through similar approaches, we revealed that the DNA binding domain of p53 and the middle region of Snail served as a binding domain (Figures 4, D–F, and W4, E and F).

Figure 4
Direct binding of Snail and p53. (A) Immunoprecipitation between p53 and Snail. In p53-deficient cells, Snail and p53 are not detected. In contrast, Capan-1 (K-Ras-mutated cell) shows an obvious interaction between them. DU145 shows weak interaction. ...

Identification of Specific p53-Snail Binding Inhibitors

If Snail-p53 binding was essential for K-Ras-mediated p53 suppression in pathologic status, preventing p53-Snail binding would reactivate p53 under the K-Ras-activated condition and blocking p53-Snail binding would be a promising drug target. Thus, we developed the ELISA system and screened the binding blocker between p53 and Snail (Figure W5, A and B). From approximately 150 kinds of chemicals, we identified 3 as inhibitors of Snail and p53 (Figure W5C). These chemicals showed a dose-dependent inhibition of Snail and p53 binding (Figures 5A and W6A). To confirm this, we performed the GST pull-down and measured the expression of p53 and its targets after treatment of these chemicals. All of them could block the interaction of p53 and Snail and induce p53 expression (Figures 4B and W6B). We could also observe the induction of p53 up-regulated modulator of apoptosis and p21 by treatment of these chemicals (Figure 5C). A more interesting feature was that induction of p53 was detected only in K-Ras-mutated cells but not wild type K-Ras-harboring cells (Figure 5D). The similar structure of quercetin and morin suggested that our screening system was reliable (Figure W5C). Although these chemicals were isolated from natural compounds, they would serve as an initial compound for generating an anticancer drug through sequential modification. Finally, we checked the effect of our chemicals on Snail-mediated p53 suppression. p53 reduction by cotransfection of Snail was blocked by the treatment of chemicals by nos. 3 and 9 (Figure 5, E and F). These results suggested that blocking of p53-Snail interaction could restore the p53 expression. We also examined the effect of these chemicals on cell proliferation using tryphan blue staining. These chemicals could obviously suppress cell proliferation in A549, whereas they did not show an antiproliferating effect on MKN-45 (K-Ras wild type cell; Figure W6C). In addition, ferulic acid could evoke cell death in K-Ras-mutated cells (Figure W6D). Moreover, quercetin (no. 2) was identified as an inhibitor of Snail-p53 interaction. The antitumoral effect of this chemical has been proposed in epidemiological studies. In particular, this chemical showed a prevention effect on pancreatic cancer [37,38]. This fact is consistent with our result because oncogenic mutation of K-Ras is frequently detected in those types of cancer.

Figure 5
Blocking of Snail-p53 binding induces p53 function in K-Ras—mutated cells. (A) Dose effect of chemical on His-p53-Snail binding. Increase of the OD value at a high concentration of no. 9 would be due to color of this chemical. The IC50 value of ...

Discussion

In this study, we showed that K-Ras could suppress p53 function (Figure 6). K-Ras is frequently mutated in human cancer. In pancreatic cancer, more than 80% of cancers and adenomas possess or are initiated by the active mutation of K-Ras [2,4]. In contrast, the H- or N-Ras mutation rate is relatively lower than that of K-Ras [2], suggesting that the tumorigenic property or tumor-promoting force of K-Ras may be better than that of other Ras. However, the MAPK-activating or cell-proliferating ability of three kinds of Ras is known to be similar to each other [1,3]. These facts imply that there is an additional and unique function of K-Ras that may contribute to tumor progression. On the basis of our result, we propose that p53 suppression is the unique function of oncogenic K-Ras. According to previous observations, elimination of oncogenic K-Ras can induce p53 despite the mutant p53 cell line [39]. These results are exactly consistent with our result that oncogenic K-Ras can suppress wild type and mutant p53 (Figure 1, F and G).

Figure 6
Diagram for summary. When K-Ras is activated by a genetic mutation, ATR is activated and promotes p53 expression. Conversely, ATR induces Snail and suppress p53. Despite this balancing, mutant K-Ras can escape from p53-induced growth suppression and drive ...

We also reveal that K-Ras-mediated p53 suppression is achieved through Snail (Figures 2A and and6).6). Snail has been known as a transcriptional suppressor and a key regulator during embryonic development (cell migration and EMT). Owing to its EMT-promoting function, overexpression of Snail is frequently detected in advanced cancer or metastatic carcinoma [23,35–37]. However, as shown in Figure 3F and other's report [25], protein half-life of Snail is too short to perform the transcriptional repression. In fact, we did not observe the obvious reduction of E-cadherin, a well-known target gene of Snail [36], by overexpression of Snail (data not shown). Therefore, we assume that the rapid turnover of Snail is related with its oncogenic function. In fact, under the presence of p53, Snail expression is more significantly reduced (Figure 2F). This result indicates that posttranslational reduction or low expression of Snail in cell, despite the sufficient mRNA expression (Figure 2D), would be related with p53 reduction. Our result may explain why Snail overexpression shows the reoccurrence of tumor from ionizing radiation (IR) and chemotherapy [37,40]. For the successful reoccurrence, tumor should obtain the resistance to DNA damage-induced apoptosis. Thus, the overexpression of Snail may render the resistance to DNA damage through p53 suppression.

Until now we do not know the detailed mechanism of the p53 reduction by Snail. Although we have checked several kinds of possibilities including ubiquitin-mediated degradation, transcriptional repression, and shortening of p53 half-life (Figure W2), we did not obtain a clear mechanism. These negative results suggest that a novel p53 suppression mechanism would be present in the Snail-mediated p53 reduction. In fact, we recently observed the p53 in vesicle-like structure in Snail-transfected cells (data not shown). This feature may provide the clue to reveal the p53 suppression mechanism.

We also reveal that Snail is obviously induced by ATR activity (Figure 3C). Because ATR is activated by DNA damage [41], IR or chemotherapy may also induce Snail, resulting in the suppression of p53. Although IR mainly activates ATM [42], several kinds of literatures show the induction of ATR. Thus, activation of ATR, despite p53 induction ability through Chk1-mediated phosphorylation [41,42], may confer the possibility of reoccurrence. In fact, elimination of si-Snail can extend UV-mediated p53 activation (our unpublished data; data now shown).

In this study, we reveal that p53 and Snail form the very strong complex that promotes reduction of both proteins. Their binding is achieved through DNA binding domain of p53 and middle region of Snail (Figure 4F). However, why their binding has not been detected for a long time is mysterious. Concerning this, we assume that owing to the rapid elimination of interacted p53 and Snail, their binding has not been easily detected (Figures W3C and and3F3F).

Thus, it is very difficult to prove that Snail suppresses p53 through direct interaction in response to oncogenic K-Ras because of the rapid disappearance of p53 and Snail complex. Therefore, we tried to identify the p53-Snail binding inhibitor. If the inhibitor blocks the p53-Snail binding, p53 will be stabilized or increased in K-Ras-mutated cell. In contrast, normal cells will not induce p53 because p53 is not eliminated by K-Ras or Snail. As described, we have generated the ELISA-based screening system and isolated three compounds (Figure W5). Interestingly, two chemicals (quercetin and morin) have a very similar structure. In our previous study, we have screened the p53 activator in K-Ras-mutated cells through Western blot-based assay system (our submitted data). From this independent analysis, quercetin has been identified as a specific activator of p53 in K-Ras-mutated cells. Considering these results, quercetin or similar chemicals would possess a p53-inducing ability through blocking of Snail-p53 binding.

Although quercetin or its derivatives are natural compounds and show broad biologic effect, it may be possible to generate a specific inhibitor against Snail-p53 binding through further modification or development. Blocking of Snail-p53 binding induces p53 in a K-Ras-dependent manner (Figure 5) strongly suggests that oncogenic K-Ras suppresses p53 through Snail-p53 binding. Moreover, our chemicals can induce p53 target genes (Figure 5F) implying that they can serve as a basic platform for specific anticancer drug development. Recently, we are performing a second chemical screening and getting more effective compounds that also show K-Ras-specific p53 induction at 0.5-µM ranges.

Our results indicate that oncogenic K-Ras blocks the p53 activation through the induction of Snail, which suppresses p53 through direct interaction (Figure 6). This is achieved through K-Ras-mediated ATR activation that has been previously reported [31]. On the basis of this mechanism, cancer cells may escape from p53-induced apoptosis and senescence despite K-Ras mutation.

Taken together, our novel pathway, that is, Snail-mediated p53 suppression, would be a useful target for anticancer drug development, in particular, K-Ras-mutated cancers such as pancreatic, lung, and colon cancers.

Acknowledgments

We thank Aging Tissue Bank in Pusan National University for supplement of natural compounds.

Footnotes

1This work was supported by the Bio-Scientific Research Grant funded by the Pusan National University (PNU, Bio-Scientific Research Grant; PNU-2008-0596-000) and by a research grant for the Research Team for Longevity Life Sciences in Pusan National University.

2This article refers to supplementary materials, which are designated by Figures W1 to W6 and are available online at www.neoplasia.com.

Supplementary Material

Supplementary Figures and Tables:

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