Introduction

A majority of clinical specimens from mesothelioma patients showed deletion of INK4A and ARF regions with wild-type p53 genotype [1]. The deletion results in loss of the p14ARF and p16INK4A gene, and is consequently associated with an uninhibited activity of MDM2 which ubiquitinates p53 and promotes the degradation process. Mesothelioma is therefore functionally defective of p53 functions despite bearing wild-type p53 genotypes. Activation of the suppressed p53 functions can be one of the therapeutic strategies for mesothelioma patients who are currently treated with DNA damaging agents since p53 facilitates apoptotic cell death [2]. Recent whole-exome sequencing data with the clinical specimens confirmed high frequency of the INK4A and ARF deletion and also revealed another common mutation at the neurofibromatosis type 2 (NF2) gene [3, 4]. The genetic mutation and aberrations downstream to NF2 resulted in a loss of functions of the Hippo pathway. The defective Hippo pathway influenced a number of cellular functions, which included increased activity of focal adhesion kinase (FAK) [5]. A relationship between of NF2 expression levels and NF2 mutation however remained unknown.

A small molecule targeting p53 is one of the therapeutic options for cancer. CP-31398 was initially designed to induce structural change of mutated p53 and to restore the functions [6, 7]. Subsequent studies however demonstrated that CP-31398 did not bind to p53 although the agent augmented expression of wild-type p53 [8, 9]. Different studies also suggested that the agent was inhibitory to p53 ubiquitination and stabilized wild-type p53 [10]. Nevertheless, the precise mechanism of augmenting p53 was not fully characterized and can be subjected to other genetic backgrounds which influence p53 functions and the expression levels. In contrast, nutlin-3a which blocked the binding of MDM2 to p53, suppressed MDM2-mediated p53 ubiquitination process, prolonged half-life of p53 and up-regulated the expression [11]. Both CP-31398 and nutlin-3a increase endogenous p53 levels and can thereby contribute to cell death although the mechanism of up-regulating p53 may not the same. CP-31398 was not examined for the growth suppressive activities in mesothelioma and nutlin-3a-mediated augmentation of p53 in mesothelioma was not well investigated [12]. On the other hand, several FAK inhibitors were tested for anti-tumor effects in mesothelioma and produced cytotoxic effects greater on mesothelioma with decreased NF2 expression than on those with unimpaired expression [13]. A FAK inhibitor was further investigated in a clinical study for the safety and feasibility [14].

Interactions between p53 and FAK were not well investigated, but enhanced FAK activity suppressed p53 expression in part due to inhibiting MDM2 functions [15, 16]. FAK phosphorylated at tyrosine 397 was a marker for FAK activation and the phosphorylation increased MDM2 activity, which subsequently facilitated p53 ubiquitination. In addition, FAK could be physically associated with p53 and block the transcription [17]. In contrast, p53-mediated regulation of FAK activity was scarcely reported. A previous study however indicated that wild-type p53 but not mutated p53 down-regulated FAK transcripts probably through binding of p53 to the FAK regulatory region [18]. These data collectively suggest that up-regulated FAK expression decreases p53 and the down-regulated p53 further augmented FAK expression.

In the present study, we examined growth suppressive activity of CP-31398 and nutlin-3a in mesothelioma with the wild-type or mutated p53 genotype. The present study also investigated a combinatory role of both agents in the growth suppression and tested possible combinatory effects of CP-31398 and a FAK inhibitor.

Materials and methods

Cells and agents

Human mesothelioma cell lines, MSTO-211H, NCI-H28, NCI-H226, NCI-H2052 and NCI-H2452, and mesothelial Met-5A cells which were immortalized with p53-inactivating SV40 T antigen, were purchased from American Type Culture Collection (Manassas, VA, USA). JMN-1B, EHMES-1 and EHMES-10 cells, established from Japanese patients, were kindly provided by Dr. Hironobu Hamada (Hiroshima University, Hiroshima, Japan) [19]. MSTO-211H, NCI-H28, NCI-H226, NCI-H2052, EHMES-10 and NCI-H2452 cells had wild-type p53 genotype but NCI-H2452 cells expressed truncated p53 protein [20]. JMN-1B (G245S) and EHMES-1 (R273S) had mutated p53 genotype. All the p53 wild-type mesothelioma cells were defective of p14 and p16 expressions because of either loss of the transcription due to methylation in the regulatory regions or deletion of the genomic DNA. CP-31398, nutlin-3a and defactinib were purchased from Tocris Bioscience (Bristol, UK), ChemieTek (Indianapolis, IN, USA) and Selleck Chemicals (Houston, TX, USA), respectively.

In vitro cytotoxicity

Cells (2 × 103/well) were seeded in 96-well plates and were treated with different concentrations of the agent. Cells were cultured for 4 days and the viability was determined with a colorimetric cell-counting WST kit (Wako, Osaka, Japan) (WST assay). The amount of formazan produced from WST-8 reagent was determined with the absorbance at 450 nm and the relative viability was calculated based on the absorbance without any treatments. Combinatory effects and a half maximal inhibitory concentration (IC50) values were estimated with CalcuSyn software (Biosoft, Cambridge, UK) based on the WST assay. Combination index (CI) values at respective fractions affected (Fa) points showed relative levels of suppressed cell viability. CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic actions, respectively. Cell numbers were also counted with the trypan blue dye exclusion assay.

Cell cycle analysis

Cells treated with an agent for 2 days were fixed in ice-cold ethanol, incubated with RNase (50 µg/ml) and stained with propidium iodide (50 µg/ml). The staining profiles were analyzed with FACSCalibur (BD Biosciences, San Jose, CA, USA) and CellQuest software (BD Biosciences).

Western blot analysis

Cell lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nylon filter and then reacted with antibody against phosphorylated p53 at Ser 15 (catalog number: #9284) or 46 (#2521), p21 (#2947), caspase-3 (#9668), cleaved caspase-3 (#9661), cleaved caspase-8 (#9496), cleaved caspase-9 (#9505), PARP (which also detects cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr397 (#3283), AMPKα (#2532), phosphorylated AMPKα (Thr172) (#2535), phosphorylated MDM2 (Ser166) (#3521), CHK2 (#2662), ATR (#2790), phosphorylated CHK1 (Ser345) (#2348), phosphorylated CHK2 (Thr68) (#2661), (Cell Signaling, Danvers, MA, USA), p53 (Ab-6, Clone DO-1), phosphorylated ATR (Ser428) (Ab-2607920) (Thermo Fisher Scientific, Fremont, CA, USA), MDM2 (sc-965), CHK1 (sc-8408) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ubiquitin (ab7780), phosphorylated KAP1 at Ser 824 (ab70369), phosphorylated ATM (Ser1981) (ab81292) (Abcam, Cambridge, MA, USA), ATM (07-1286) (Millipore, Temecula, CA), phosphorylated H2AX at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), and actin (#4970) (Cell Signaling) as a loading control followed by appropriate second antibody. The membranes were incubated with the ECL system (GE Healthcare, Buckinghamshire, UK) and imaged with ImageQuant LAS 4000 (GE Healthcare).

Statistical analysis

ANOVA test was used to statistically analyze data.

Results

Growth inhibitory effects of CP-31398

We examined CP-31398-mediated growth inhibition with human mesothelioma with wild-type and mutated p53 genotypes (Fig. 1a). We classified NCI-H2452 and Met-5A cells as a p53-mutated group since NCI-H2452 cells expressed truncated p53 protein [20] and Met-5A expressed p53-inactivating SV40 T antigen. A colorimetric assay with the WST reagent showed that CP-31398 produced the inhibitory effects on mesothelioma and the IC50 values were not different among the cells with regard to the p53 genotype (average IC50 value ± SE 7.51 ± 1.06 µM for cells with the wild-type p53, 5.57 ± 0.11 for those with the mutated p53 group) (P = .15). We also tested the growth inhibitory effects with a dye exclusion assay (Fig. 1b). Cells treated with CP-31398 at 3 µM showed growth retardation and those treated with a higher concentration decreased cell numbers. These data collectively indicated that CP-31398 produced anti-tumor effects on mesothelioma in a p53-independent manner.

Fig. 1
figure 1

Growth inhibitory activity and molecular expression induced by CP-31398 in mesothelioma. a Cells were treated with various concentrations of CP-31398 for 4 days and the cell viabilities were measured with a colorimetric WST agent. Relative viability was calculated based on untreated cells. IC50 values calculated with CalcuSyn software are shown. Averages and SE bars are shown (n = 3). b Live cell numbers after treated with CP-31398 were counted with a trypan blue dye exclusion assay. Averages and SE bars are shown (n = 3). c Cells were treated with CP-31398 for 24 h and the expression of respective molecules as indicated was analyzed with Western blot analysis. An upper and a lower arrow indicate authentic and truncated p53, respectively. Actin was used as a loading control

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Increased endogenous p53 expression in CP-31398-treated cells

We then examined whether CP-31398 augmented expression levels of p53 (Fig. 1c). CP-31398 treatments increased the p53 levels in wild-type p53 cells except EHMES-10 cells, whereas the p53-mutated group showed inconsistent results. NCI-H2542 cells showed decreased p53 of a truncated form, and JMN-1B cells came to express 2 kinds of the molecules at 53 kDa and a lower molecular size. EHMES-1 cells up-regulated the p53 expression but Met-5A cells remained unchanged for the expression. Expression of p21, one of the p53 targets, was temporally up-regulated in mesothelioma with wild-type p53 and NCI-H2452 cells, whereas the p53-mutated cells rather down-regulated the p21 levels. Previous study showed that CP-31398 could activate the AMP-activated protein kinase (AMPK) pathway through up-regulated phosphorylation of AMPK [21], and that the enhanced AMPK activity increased p21 expression [22]. The current study showed increase of AMPK or phosphorylated AMPK in NCI-H2052 and EHMES-10 cells but not in others among the wild-type p53 cells, and decrease of that in NCI-H2452 cells among the p53-mutated group, indicating that CP-31398-induced p21 expression was not attributable to AMPK regulation. These data also indicated that CP-31398 augmented p53 levels in mesothelioma with the wild-type p53 but the growth suppressive effects was irrelevant to p53 up-regulation.

We also examined expression of MDM2 and the phosphorylation at Ser166, an activated marker of MDM2, and ubiquitinated protein levels in cells treated with CP-31398 (Fig. 2). MDM2 expression and/or the phosphorylation increased with CP-31398 in the wild-type p53 cells although EHMES-10 poorly expressed MDM2 (Fig. 2a). In contrast, responses of mutated p53 cells to CP-31398 were inconsistent. MDM2 and the phosphorylation levels decreased in NCI-H2452, but increased in MET-5A cells. JMN-1B cells temporally up-regulated the expression, but EHMES-1 cells showed mixed responses, increased MDM2 but decreased the phosphorylation. We also examined levels of MDM2 phosphorylation in wild-type p53 cells treated with nutlin-3a, a MDM2 inhibitor, and showed that the phosphorylation increased with nutlin-3a (Fig. 2b). Nutlin-3a-mediated changes of the expression were due to a reciprocal inhibition between p53 and MDM2. Nutlin-3a inhibited MDM2 functions and increased p53 levels, and then the augmented p53 consequently increased MDM2 levels. The increased MDM2 and the phosphorylation in wild-type p53 cells treated with CP-31398 suggested that CP-31398 could inhibit MDM2 and increase p53 levels like nutlin-3a.

Fig. 2
figure 2

CP-31398 or nutlin-3a induced differential expression of MDM2 and ubiquitination. Cells were treated with CP-31398 (a) or nutlin-3a (b) for 24 h and the expression of respective molecules as indicated was analyzed with Western blot analysis. Actin was used as a loading control (b)

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We further investigated ubiquitination levels induced by CP-31398 (Fig. 2). The ubiquitinated protein levels were down-regulated in wild-type p53 cells except NCI-H226 and NCI-H2052 cells which rather increased the ubiquitination (Fig. 2a). In contrast, ubiquitination changes in mutated p53 cells were inconsistent, decreased in NCI-H2452 and EHMES-1 cells but increased in JMN-1B and MET-5A cells. We also examined ubiquitination by nutlin-3a and showed that nutlin-3a decreased ubiquitination in MSTO-211H and NCI-H28 cells (Fig. 2b). These data suggested that CP-31398-mediated increase of wild-type p53 was also attributable to a non-ubiquitination process.

Growth inhibitory effects of nutlin-3a

We also examined nutlin-3a-mediated growth inhibition in mesothelioma with the colorimetric assay (Fig. 3a). Sensitivity to nutlin-3a was greater in cells with the wild-type p53 excluding EHMES-10 cells (average IC50 ± SE: 3.64 ± 2.87 µM) than in those with the p53-mutated group (30.49 ± 7.93) (P < .01). A mechanism of resistance to nultin-3a in EHMES-10 remained uncharacterized but they expressed MDM2 at a low level (Fig. 2a). We then examined p53 expression after nutlin-3a treatments in representative mesothelioma cells (Fig. 3b). Mesothelioma with the wild-type p53 up-regulated p53 and the phosphorylation at Ser 15, whereas those of mutated p53 to a lesser extent augmented p53 and the phosphorylation. We also examined expression levels of FAK and phosphorylated FAK at Tyr 397 and found that nultin-3a did not influence FAK levels except JMN-1B cells which showed down-regulated expression, but suppressed the phosphorylation except NCI-H28 cells which did not change the level. The nutlin-3a-mediated down-regulation of phosphorylated FAK was therefore not restricted in wild-type p53 cells.

Fig. 3
figure 3

Growth inhibitory activity and molecular expression induced by nutlin-3a in mesothelioma. a Cells were treated with various concentrations of nutlin-3a for 4 days and the cell viabilities were measured with a colorimetric WST agent. Relative viability was calculated based on untreated cells. IC50 values calculated with CalcuSyn software are shown. Averages and SE bars are shown (n = 3). b Cells were treated with nutlin-3a for 24 h and the expression of respective molecules as indicated was analyzed with Western blot analysis. Actin was used as a loading control

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Combination of CP-31398 and nutlin-3a produced synergistic growth inhibition in the p53 wild-type cells

We next examined combinatory effects of CP-31398 and nutlin-3a on mesothelioma with the wild-type and mutated p53 genotype (Fig. 4). CP-31398-mediated growth suppression was further enhanced in the combination with nutline-3a and the CI values showed that the combination achieved synergistic effects in mesothelioma with the wild-type p53 (Fig. 4a). In contrast, mesothelioma with mutated p53 required did not produce synergistic but rather antagonistic effects in a majority of Fa points under a high concentration of nutlin-3a which inhibited the cell growth. We also counted live cell numbers of mesothelioma with the wild-type p53 and showed that the combinatory use of CP-31398 and nutlin-3a suppressed the cell growth greater than a treatment with the single agent (Fig. 4b). We tested cell cycle progression after the treatments (Fig. 4c; Table 1). CP-31398 increased S-phase and G2/M-phase populations, and the combination augmented sub-G1 populations in MSTO-211H cells. In contrast, the combination in NCI-H28 cells did not increase sub-G1 fractions in the combination although CP-31398 augmented G2/M-phase and nutlin-3a slightly enhanced sub-G1 populations. These data suggested that the combination induced cell death in MSTO-211H cells and cell cycle arrest in NCI-H28 cells.

Fig. 4
figure 4

Growth inhibition caused by a combinatory use of CP-31398 and nultin-3a. a Cells were treated with various concentrations of CP-31398 and nutlin-3a with an indicated concentration, and the cell viabilities were measured with a colorimetric WST agent. Relative viability was calculated based on untreated cells. Averages and SE bars are shown (n = 3). CI values in the combination were calculated with CalcuSyn software at various Fa points. b Live cell numbers after treated with CP-31398, nutlin-3a or the combination were counted with a trypan blue dye exclusion assay. Averages and SE bars are shown (n = 3). Asterisks showed P < .01. c Representative profiles of cell cycle distributions after treated with CP-31398 (15 µM), nutrin-3a (10 µM) or the combination for 48 h

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Table 1 Cell cycle distribution of mesothelioma cells treated with CP-31398, nutlin-3a or the combination
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We also examined DNA damage responses with phosphorylated H2AX at Ser 139 and KAP1 at Ser 824 in cells treated with CP-31398, nutlin-3a and the combination (Fig. 5). CP-31398 augmented phosphorylated H2AX in MSTO-211, EHMES-1 cells and to a lesser extent in JMN-1B cells, but rather decreased in NCI-H28 cells. CP-31398-induced KAP1 phosphorylation in those cells was similar to that of the H2AX phosphorylation. Increased KAP1 phosphorylation was detected in MSTO-211H, EHMES-1 and JMN-1B cells but the phosphorylation in NCI-H28 remained unchanged. In addition, DNA damage responses induced by CP-31398 was further up-regulated by nutlin-3a not only in MSTO-211H cells but also in mutated p53 cells. NCI-H28 cells did not show the augmented expression of phosphorylation. These data indicated that DNA damage responses could linked with S-phase arrest but were not associated with synergistic growth inhibition.

Fig. 5
figure 5

DNA damage responses induced by CP-31398 and combination with nutlin-3a. Cells were treated with the agent at the indicated concentration for 24 h and were subjected to Western blot analysis as indicated. Actin was used as a loading control

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We further investigated a possible involvement of ATR/CHK1 or ATM/CHK2 pathway in the DNA damage responses (Fig. 5). We examined phosphorylation of the molecules in the both pathways and calculated the relative phosphorylation levels (Supplementary Table 1). A ratio of CHK1 phosphorylation increased in MSTO-211H and EHMES-1 cells treated with CP-31398 and nutlin-3a, but the combination did not significantly augment the CHK1 phosphorylation. NCI-H28 and JMN-1B cells did not show the differential phosphorylation. We found that CHK1 expression itself decreased in the treated cells except JMN-1B cells but the mechanism remained unknown. ATR phosphorylation increased in EHMES-1 cells treated with CP-31398, nutlin-3a and the combination, but the phosphorylation in MSTO-211H cells did not in the combination. The phosphorylation in NCI-H28 cells was down-regulated with CP-31398 and nutlin-3a and that in JMN-1B remained unchanged. Expression of ATR was up-regulated in MSTO-211H cells treated with the combination, but the mechanism was unknown. These data collectively suggested that EHMES-1 cells activated the ATR/CHK1 pathway but contribution of the pathway to drug-induced DNA damages was minimal in other cells. CHK1 phosphorylation was observed in the combination-treated and nutlin-3a-treated MSTO-211H and NCI-H28 cells, indicating that DNA damage responses augmented by the combination was irrelevant to CHK1 phosphorylation. On the other hand, the CHK2 phosphorylation ratio increased in MSTO-211H, EHMES-1 and JMN-1B cells treated with CP-31398 and the combination, and in NCI-H28 cells treated with the combination. Expression of CHK2 was also down-regulated in MSTO-211H, NCI-H28 and EHMES-1 cells like CHK1 expression. ATM phosphorylation was however not correlated with the enhanced CHK2 phosphorylation except in EHMES-1 cells treated with the combination. These data implied that CHK2 phosphorylation contributed to CP-31398-induced DNA damage responses and to nutlin-3a-mediated damage augmentation, but the phosphorylation was not associated with ATM activation except EHMES-1 cells.

Combination of CP-31398 and nultin-3a increased p53 levels and suppressed FAK phosphorylation

We investigated molecular events regarding growth inhibition in MSTO-211H and NCI-H28 cells with Western blot analysis (Fig. 6). The combination of CP-31398 and nutlin-3a increased p53 and the phosphorylation levels greater than a treatment with either CP-31398 or nutlin-3a. Expression of p21 and MDM2, both of which were p53 target molecules, were consequently up-regulated in the combination. MSTO-211H cells treated with CP-31398 showed increase of cleaved PARP levels but did not further augment the levels in the combination. Expression of caspase-3 increased in MSTO-211H cells treated with nutlin-3a and the combination, and that of cleaved caspase-3 was minimally augmented in those treated with the combination. Cleavage of caspase-8 and to a lesser extent caspase-9 increased in MSTO-211H treated with the combination. These data collectively indicated that the combination activated the p53 pathway and induced apoptosis in MSTO-211H cells. NCI-H28 cells however showed different responses to the agents. Expression of cleaved PARP, caspase-3 and cleaved caspase-3 increased in NCI-H28 cells treated with nultin-3a and the combination, but cleavage of caspase-8 and − 9 was not augmented in those treated with the combination. These data suggested that the combinatory use of both agents in NCI-H28 activated p53 pathway but only induced cell growth arrest as demonstrated in cell cycle analysis.

Fig. 6
figure 6

Molecular expression in cells treated with combination of CP-31398 and nutlin-3a. Cells were treated with the agent at the indicated concentration for 24 h and were subjected to Western blot analysis as indicated. Arrows indicated PARP, cleaved PARP, cleaved caspase-8 and cleaved capase-9, and actin was used as a loading control

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We also examined whether p53 augmentation influenced FAK and the phosphorylation levels. Expression of FAK increased in nutin-3a-treated and the combination-treated MSTO-211H cells, but that of FAK remained unchanged in NCI-H28 cells. The FAK phosphorylation was down-regulated in CP-31398- or nutlin-3a-treated MSTO-211H cells and further decreased in the combination. Decrease of FAK phosphorylation was marginal in CP-31398- or nutlin-3a-treated NCI-H28 cells but significant in the combination. These data indicated that p53 did not influence FAK expression but inactivated FAK activity, and suggested that FAK inactivation contributed to the combination-induced growth inhibitory effects.

Inhibition of FAK augmented CP-31398-mediated growth effects

We examined whether FAK inhibition achieved growth suppressive effects in combination with CP-31398. MSTO-211H and NCI-H28 cells were treated with defactinib, a FAK inhibitor, and various concentrations of CP-31398 (Fig. 7a). The combination produced synergistic growth inhibitory effects with CI values less than 1. We also investigated molecular events induced by the treatments with Western blot analysis (Fig. 7b). Defactinib did not influence FAK expression but suppressed FAK phosphorylation. Combination of CP-31398 and defactinib further down-regulated phosphorylated FAK in MSTO-211H cells and decreased FAK expression in NCI-H28 cells. Defectinib-treated cells increased p53 expression and the combination-treated cells further augmented p53 and the phosphorylation levels. These data indicated that FAK inhibition produced synergistic growth suppressive effects with CP-31398 and suggested that FAK inactivation induced p53 activation.

Fig. 7
figure 7

Growth inhibition and molecular expression caused by a combinatory use of CP-31398 and defactinib. a Cells were treated with various concentrations of CP-31398 and defactinib with the indicated concentration, and the cell viabilities were measured with a colorimetric WST agent. Relative viability was calculated based on untreated cells. Averages and SE bars are shown (n = 3). CI values in the combination were calculated with CalcuSyn software at various Fa points. b Cells were treated with the agent at the indicated concentration for 24 h and were subjected to Western blot analysis as indicated. Actin was used as a loading control

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Discussion

We demonstrated in the present study that CP-31398 augmented endogenous p53 levels and a combinatory use of CP-31398 and nutlin-3a or defactinib achieved synergistic growth inhibitory effects in mesothelioma with wild-type p53 genotypes. Moreover, nutlin-3a which increased endogenous p53 expression down-regulated FAK phosphorylation, and defactinib which dephosphorylated FAK induced up-regulated p53 levels. The current study firstly reported to our knowledge anti-tumor effects of CP-31398 in mesothelioma and synergistic combinatory effects by CP-31398 and an MDM2 inhibitor or a FAK inhibitor.

CP-31398 was initially studied as an agent to structurally convert mutated p53 to wild-type p53, but the precise mechanism how CP-31398 restored p53 functions in mutated p53 cells was not well understood [23, 24]. The present study showed that CP-31398 increased p53 levels in mesothelioma with the wild-type p53 but the growth suppressive activity was irrelevant to p53 genotype. Furthermore, CP-31398-mediated inhibitory effects judged by IC50 values were similar among mutated p53 cells but the p53 responses were inconsistent in the present study. On the other hand, CP-31398-mediated actions could be similar to nutlin-3a in some of wild-type p53 cells which increased MDM2 phosphorylation and decreased ubiquitinated protein levels as found in nutlin-3a-treated cells. Nevertheless, other wild-type p53 and mutated p53 cells showed different responses to CP-31398 regarding ubiquitination. In fact, we previously showed that ubiquitinated protein levels were linked with p53 expression [25]. These data collectively indicated that CP-31398 could inhibit MDM2 activities but a non-MDM2-mediated ubiquitination pathway was also involved in the action mechanism of CP31398. In addition, the p53 mutation site of EHMES-1 cells at codon 273 was one of the sites which CP-31398 could restore the p53 functions [10]. The mutated sequence in EHMES-1 cells (R273S) was different from the sequence in a previous study (R273H) which demonstrated p53 activation with CP-31398 [10], but CP-31398-treated EHMES-1 cells up-regulated p53 expression as found in wild-type p53 mesothelioma. Nevertheless, CP-31398 down-regulated p21 expression in EHMES-1 cells in contrast to the up-regulation in wild-type p53 cells, which indicated that p53 functions were not restored in EHMES-1 cells. The CP-31398-mediated p21 response was not regulated by p53 expression as also demonstrated in EHMES-10 and NCI-H2452 cells which showed p21 up-regulation under p53 down-regulation. Previous studies also reported that p21 augmentation by CP-31398 was irrelevant to p53 functions but the mechanism of p53-independent p21 up-regulation remained uncharacterized [9, 26]. We recently found that the CP-31398-mediated p21 augmentation was in part regulated by a transcriptional factor YY1 which controlled a number of gene expression [27]. Nutlin-3a augmented p21 expression even in p53-muated cells in contrast to CP-31398 which down-regulated the expression, indicating a mechanism to regulated p21 expression was different between CP-31398 and nutlin-3a. We also showed that CP-31398 induced DNA damage responses irrespective of the p53 genotypes but the responses were irrelevant to AMPK phosphorylation.

FAK plays a certain role in regulation of p53 expression through MDM2, which was shown in previous studies that decreased FAK expression with the shRNA augmented p53 levels by phosphorylated MDM2 [15, 16]. The present study demonstrated that defactinib decreased FAK phosphorylation and increased p53 in wild-type p53 mesothelioma. Furthermore, CP-31398 suppressed FAK phosphorylation without decrease of FAK expression. A combinatory use of defactinib and CP-31398 consequently down-regulated FAK phosphorylation, and p53 and the phosphorylation levels was up-regulated greater than a treatment with the single agent. These data indicated that inhibition of FAK activity led to p53 activation and suggested that tumors with poor interactions with extracellular matrix, which came to decrease FAK activity, suppressed the cell proliferation or were prone to trigger cell death mechanism due to p53 activation. On the other hand, the present study also showed that p53 up-regulation down-regulated FAK activity. Mesothelioma cells treated with CP-31398 or nutlin-3a suppressed FAK phosphorylation with minimally influencing FAK expression, and the combination of CP-31398 and nutlin-3a further inhibited the FAK phosphorylation. A mechanism of p53-mediated regulation of FAK phosphorylation was not well understood. A p53 binding site was identified in regulatory region of FAK gene and p53 possibly inhibited FAK transcripts [18], but the preset study showed that p53 down-regulated FAK phosphorylation but not FAK expression. A recently study however showed that wild-type p53 but not mutated p53 blocked FAK phosphorylation though TGF-β signaling and reactive oxygen species generated [28]. The precise mechanism how TGF-β signaling dephosphorylated FAK was currently uncharacterized, but we presume that up-regulated p53 decreased tumor cell growth and the cells might shut off a growth signal from extracellular matrix, which resulted in dissociation of FAK from cell membrane and consequently in FAK dephosphorylation. The present study therefore indicated that p53 expression and FAK inactivation were reciprocally correlated and we think that a precise mechanism of the reciprocal interactions between p53 expression and FAK activity is the next issue to be clarified. The present study also showed that CP-31398 and nutlin-3a or defactinib achieved combinatory effects in accordance with augmented p53 and down-regulated FAK phosphorylation, and consequently indicated that the combination effects produced with nutlin-3a or defactinib were attributable to both decreased FAK activity and increased p53 levels. A previous study indicated that FAK inhibitor was more effective to NF2-low cells than NF2-high cells [13] but our preliminary data however showed that sensitivity to defactinib was irrelevant to NF2 expression and to p53 genotype. We presume that targeting p53 and NF2 in combination is one of the therapeutic strategies in terms of mesothelioma genetics but detailed analysis on the mechanism of the drug actions is required.

We noticed differential responses of MSTO-211H and NCI-H28 cells to nutlin-3a and the combination with CP-31398. A treatment with nutlin-3a decreased FAK phosphorylation in MSTO-211H but not in NCI-H28 cells, and the combinatory treatment with CP-31398 induced apoptosis in MSTO-211H cells but cell cycle arrest in NCI-H28 cells. Cell cycle profiles and cleavages of PARP, caspase-8 and -9 showed such discrepant responses, which might be attributable to relative insensitivity of NCI-H28 cells to the agents in comparison with MSTO-211H cells. Moreover, we showed that DNA damage responses was induced in MSTO-211H cells and the responses were augmented by nuttlin-3a. NCI-H28 cells however did not show the enhanced responses with CP-31398 or the combination. Nevertheless, DNA damage responses were also induced in mutated p53 cells and we examined the combination effects with mutated p53 cells in terms of caspase cleavages (Supplementary Fig. 1). We found that CP-31398-mediated cleavages of PARP and caspase 3 were also augmented by nutlin-3. These data suggested that wild-type p53 expression augmented apoptotic pathways and contributed to synergistic cytotoxicity in the combination, but mutated p53 was rather inhibitory to the pathways. The inhibition was perhaps due to bypassing caspases-mediated pathways, and resulted in rather antagonistic action in the combination. The current investigation on DNA damage responses in terms of ATR/CHK1 and ATM/CHK2 pathways was also complex. CHK1 can be activated by CP-31398 but the activation was not directly linked with nutlin-3a-mediated augmentation of DNA damage responses. In contrast, phosphorylated CHK2 levels were up-regulated by CP-31398 and the combination with nutlin-3a. Nevertheless, the CHK2 activation was not associated with ATM phosphorylation. Cross-talk between ATR/CHK1 and ATM/CHK2 and non-ATM transducers can regulate CHK2 activation, which makes the checkpoint mechanism of cell cycle complicated and be dependent on cells tested [29]. We presume at this moment that CHK2 is more important than CHK1 in DNA damages induced by CP-31398 and in the augmentation by nutlin-3a since CHK2 increased p53 expression partly through inhibiting MDM2 functions [29].

In the present study, we showed that CP-31398 produced growth inhibitory effects on mesothelioma in p53-independent manner and achieved synergistic combinatory effects with nutlin-3a or defactinib. We furthermore demonstrated that p53 expression and FAK phosphorylation were reciprocally regulated and that the combinatory effects were associated with p53 up-regulation and FAK dephosphorylation. An MDM2 inhibitor and a FAK inhibitor is a potentially therapeutic agent for mesothelioma and a combinatory use of the inhibitors and a p53-activating agent is a therapeutic option for mesothelioma.