Abstract
Background:
Malignant pleural mesothelioma (MPM) is a highly aggressive tumour that is first-line treated with a combination of cisplatin and pemetrexed. Until now, predictive and prognostic biomarkers are lacking, making it a non-tailored therapy regimen with unknown outcome. P53 is frequently inactivated in MPM, but mutations are extremely rare. MDM2 and P14/ARF are upstream regulators of P53 that may contribute to P53 inactivation.
Methods:
A total of 72 MPM patients were investigated. MDM2 immunoexpression was assessed in 65 patients. MDM2 and P14/ARF mRNA expression was analysed in 48 patients of the overall collective. The expression results were correlated to overall survival (OS) and progression-free survival (PFS).
Results:
OS and PFS correlated highly significantly with MDM2 mRNA and protein expression, showing a dismal prognosis for patients with elevated MDM2 expression (for OS: Score (logrank) test: P⩽0.002, and for PFS: Score (logrank) test; P<0.007). MDM2 was identified as robust prognostic and predictive biomarker for MPM on the mRNA and protein level. P14/ARF mRNA expression reached no statistical significance, but Kaplan–Meier curves distinguished patients with low P14/ARF expression and hence shorter survival from patients with higher expression and prolonged survival.
Conclusions:
MDM2 is a prognostic and predictive marker for a platin–pemetrexed therapy of patients with MPMs. Downregulation of P14/ARF expression seems to contribute to MDM2-overexpression-mediated P53 inactivation in MPM patients.
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Main
Malignant pleural mesothelioma (MPM) is a highly aggressive tumour, which is linked to prior exposure to asbestos with a latency period of 20–30 years in approximately 80% of all cases (Weill et al, 2004; Hazarika et al, 2005; Goudar, 2008). Systemic therapy represents the primary treatment option for most patients (Treasure and Sedrakyan, 2004; Tsao et al, 2009), but standard MPM therapy is still deficient and decisions for radiotherapy, surgery or combined approaches are based on a case-by-case decision leading to a palliative treatment approach for most patients (Guyatt et al, 2006; Muers et al, 2008; Stahel et al, 2009; Astoul et al, 2012). Gender, histological subtype and haematological parameters have been identified as important prognostic parameters (Flores et al, 2007; Rusch et al, 2012).
Combination of antifolates (e.g., pemetrexed) and platin derivatives is considered as the most effective regimen for MPM (Tomek and Manegold, 2004; Kindler, 2008). In that therapeutical setting, patients show response rates of approximately 40% (stable disease and partial response) with a progression-free survival (PFS) of 5.7 months (Vogelzang et al, 2003). As reason for these low response rates and short PFS, expression differences of members of the folic acid pathway, folic acid transport and activation, which are important for the uptake and metabolism of pemetrexed, were discussed controversially in the past (Righi et al, 2010; Christoph et al, 2012; Lustgarten et al, 2013; Mairinger et al, 2013a, 2013b).
Additional investigations on the gene level showed that inactivation of tumour-suppressor genes are frequent in MPM (Frew et al, 2009). In contrast to other solid tumours, mutations of the TP53 gene are extremely rare in MPM, so other mechanisms such as deletion of the locus or methylation contribute to inactivation of P53 (Papp et al, 2001a, 2001b; Toyooka et al, 2008). Numerous noxious stimuli activate the P53 protein by posttranslational modifications resulting in cell cycle arrest, cellular senescence or apoptosis (Harris and Levine, 2005).
Among other factors, P53 activity and stability is tightly controlled by the E3 ubiquitin ligase (MDM2; also HDM2). Overexpression of MDM2 in some tumour types can lead to a loss of P53 regulatory function by increased proteasomal degradation of P53 (Jones et al, 1995; Montes de Oca Luna et al, 1995; Parant et al, 2001; Marine et al, 2006; Ringshausen et al, 2006). This pathomechanism is considered to be of importance in a variety of malignant tumours, including lung, breast, colon, stomach and hepatocellular carcinomas (Toledo and Wahl, 2006). Approximately 20% of all MPM show strong nuclear MDM2 expression, restricted to epitheloid MPM or the epitheloid components of biphasic MPM, and these MDM2-positive MPM show significantly decreased overall survival (OS) (Mairinger et al, 2014).
The physiological inhibitor of MDM2 is P14/ARF, and loss of P14/ARF activity may have a similar effect as loss of P53 (Kanellou et al, 2009). P14/ARF is recognised as a tumour suppressor inducing cell cycle arrest in a P53-dependent and P53-independent manner (Huang et al, 2003; Chen et al, 2005; Miao et al, 2010). Thereby, P14/ARF may control TP53 transcription, repress P53 degradation that is not MDM2-mediated and stimulate P53 activity (Van Maerken et al, 2011). Additionally, loss of P14/ARF activity seems to occur in a reciprocal manner to P53 loss and seems to be typical for tumours that are TP53 wild type (Huang et al, 2003).
In sum, reliable predictive biomarkers in MPM are lacking. Additionally, a personalised therapeutic concept is eagerly needed.
In the present study, we sought to determine whether decreased activity of the physiological MDM2 inhibitor P14/ARF contributes to MDM2-mediated inactivation of P53 in MPM.
Materials and Methods
Patient collective
From the MPM database of the Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany, 72 formalin-fixed, paraffin-embedded (FFPE) specimens from patients harbouring a MPM were selected for quantitative real-time PCR (qPCR) and IHC analysis. Samples from 2004 to 2010 were investigated. Inclusion criteria for this study were complete data with respect to follow-up, treatment and sufficient FFPE tissue. The study design was approved by the ethical committee of the University Hospital Essen (ID: 14-5775-BO). The investigations conform to the principles of the Declaration of Helsinki.
FFPE tissue preparation was performed according to the institutional standards. The fresh tissue was fixed in 4% buffered formalin for 24 h and subsequently embedded in paraffin. For diagnostic classification, multiple 1–4-μm thick sections were used for IHC and stained with haematoxylin and eosin. The most representative part of the tumour was used for subsequent analysis.
Immunohistochemistry
Tissue microarrays were constructed from FFPE blocks. Three cores with a diameter of 0.6 mm were taken from different areas of each tumour to take possible tumour heterogeneity into account. When feasible, a core containing only normal, non-malignant pleura was taken from every specimen, which served as a negative control.
IHC for MDM2 was newly established. After validation on reference tissues (liposarcoma as a malignant mesenchymal tumour with a consistent strong expression of MDM2), the immunohistochemical investigations were performed with an antibody directed against MDM2 (clone IF2, Calbiochem, Darmstadt, Germany; dilution: 1 : 80). Protein expression was assessed using a four-stage semiquantitative IHC scoring system based on the percentage of tumour cell nuclei with a positive immunoreaction (Score 0: no immunohistochemical signal; Score 1 (weak expression): 1–25%; Score 2 (moderate expression): 26–50%; Score 3 (strong expression): >50%). MPM were only considered positive for MDM2 when a strong nuclear staining could be observed comparable to the positive controls (liposarcoma). Tumour cells with a weak immunohistochemical nuclear or only cytoplasmic signal for MDM2 were not counted.
RNA isolation and qPCR
Expression levels of ACTB (actin, beta; reference gene), MDM2 and P14/ARF were investigated by using hydrolysis probes (also known as TaqMan probes) for qPCR. Therefore, RNA was isolated from FFPE tissue. Only tumour tissue was used for mRNA extraction. This was carried out by macrodissection (cutting only the tumour region). Three-to-five sections of 4 μm were cut from one FFPE block by using a microtome (Leica, SM 2000 R, Wetzlar, Germany). Total RNA was isolated by using the miRNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, except for two modifications (proteinase K digestion overnight; elution in a final volume of 25 μl in RNAse-free water). RNA concentrations were measured using the Nanodrop UV/VIS spectrometry (NanoDrop ND-1000, PEQLAB Biotechnologie GmbH, Erlangen, Germany). RNA concentrations ranged from 73 ng μl−1 to 3789 ng μl−1. Until further use, RNA was stored at −80 °C. For cDNA synthesis, the iScript Select cDNA Synthesis Kit and protocol from Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used. Approximately 1 μg of the total RNA was used with a final concentration of 100 ng μl−1 per reaction. cDNA was stored for short term (<1 week) at 4 °C and otherwise at −20 °C.
For qPCR, the TaqMan Gene Expression Assays on Demand (AoD) for ACTB (Hs03023943_g1), MDM2 (Hs01066942_m1) and P14/ARF encoded by CDKN2A (Hs99999189_m1) were used (Thermo Fisher Scientific, Waltham, MA, USA). The primer–probe volumes were modified by using 50% of the total reaction volumes that were recommended by the manufacturer. For each reaction, 50 ng cDNA were applied. The AoD were chosen because of their short amplicon size (ACTB: 93 nt, MDM2: 89 nt and P14/ARF/CDKN2A: 72 nt) and, additionally, because they spanned exon–exon boundaries to circumvent simultaneous detection of genomic DNA. Each target was measured in triplicates for each patient. Non-template controls were processed for each AoD on each reaction plate as negative control. Table 1A summarises the applied chemicals and volumes for the qPCR and Table 1B shows the qPCR conditions. ACTB is the standard reference gene for investigation of MPM at our institution. That is based on previous experiments testing several potential reference genes using the geNorm and NormFinder algorithms. ACTB showed robust and stable expression in MPM and hence was processed for normalisation purposes and as reference gene. Ct-values of MDM2 and P14/ARF were normalised to the mean values of ACTB. Data analysis and qPCR were performed on a Roche LightCycler 480 II (Roche, Basel, Switzerland) and the corresponding software. All qPCR experiments were performed in concordance with the MIQE-guidelines (Bustin et al, 2009).
Statistical analysis
Statistical analysis was performed using the statistical computer language R (r-project.org; version R i386 2.15.1).
Gene expression analysis
Analysis of OS and PFS were done by using the proportional hazards model (also called Cox-regression (COXPH-model)), and statistical significance was determined using the likelihood ratio test, Wald test and Score (logrank) test. With respect to functional scale-differences in biological systems, proportional hazards model analysis was done in a linear and logarithmic scale for MDM2 mRNA and protein expression and for P14/ARF mRNA expression. For association between either protein expression or mRNA expression with respect to gender, patients’ age at time of diagnosis, age of the paraffin blocks and histological subtype of MPM to OS and PFS, a proportional hazards model was calculated.
OS and PFS were visualised by creating single-factorial and combined Kaplan–Meier curves (also called product limit estimator). Kaplan–Meier curves with a confidence interval of 95% (CI: 95%) were calculated based on existing survival data.
The Spearman’s rank correlation coefficient (also called the Spearman’s rho) was used to calculate correlations between the expression levels of the tested genes. Additionally, this test was also used to rule out a possible association between expression and age of the patients, age of the FFPE tissue, gender and clinical data.
The Mann–Whitney U (also called the Wilcoxon rank-sum test) was used, for example, to test associations between the mean protein expression obtained from three cores after IHC or mRNA expression and dichotomous variables (e.g., gender).
The level of statistical significance was defined as P⩽0.05.
Results
A collective of 72 patients was investigated. Sixty-five patients (90%) gave evaluable IHC data with respect to MDM2 immunoexpression. Out of the overall collective, 48 specimens were analysed via qPCR for the mRNA expression of MDM2 and P14/ARF. The relationship of the expression of the tested markers with OS and PFS was analysed. The investigated sub-collectives are summarised in Table 2.
Clinicopathological and survival data of patients from Essen
Clinicopathological analysis
Seventy-two patients harbouring a MPM were selected. Eleven female (15%) patients and 61 male (85%) patients were investigated. The mean age of the patients was 63.8 years (median age 62.6 years, range 48–80 years). For 69 out of the 72 patients, the histological MPM subtype was available. Fifty-nine patients showed an epithelioid (82%), six biphasic (8%) and four sarcomatoid (6%) MPM subtype. For three patients, the histological subtype was not available (4%). Table 2 summarises the characteristics of the investigated patient collective.
Analysis of OS
Survival data of 72 patients were available, and 59 (81.9%) were reported dead and 13 (18.1%) were still alive when survival data were assessed. Median survival was 18.5 months (mean without censored patients: 21.9 months; range: 8.8–29.9 months).
The histological subtype correlated significantly with OS (Score (logrank) test; P=0.021), with a better prognosis for patients with an epithelioid subtype (Supplementary Figure S1).
Analysis of OS with respect to MDM2 immunoexpression
IHC gave evaluable results for 65 out of the 72 investigated patients. Fifty-four patients showed an epithelioid subtype (83.1%), five biphasic (7.7%), three sarcomatoid (4.6%) and three remained inconclusive (4.6%). Fifty-one (80%) patients were reported dead, and 13 were still alive (20%). The median OS was 19.3 months (Table 2).
OS analysis showed that gender was statistically significant (Score (logrank) test; P=0.016), with shorter survival for male patients (HR: 3.35; 95% CI, range: 1.2–9.5; data not shown). Age of the patients (Score (logrank) test; P=0.15) and age of the FFPE specimens (Score (logrank) test; P=0.48) showed no statistical correlation to OS.
According to the described scoring system, 48 patients showed Score 0 (65%), 19 Score 1 (29%), 4 Score 2 (6%) and none of the patients’ specimens had a Score 3. Of note, MDM2 expression was present in epithelioid and the epithelioid component of biphasic MPM only. All MDM2-positive MPM specimens, regardless of the score, were calculated against all MDM2-negative MPM and correlated with OS. Lacking MDM2 expression significantly associated with longer OS with respect to the linear (Score (logrank) test: P<0.0001, CI: 95%, range: 2.5–7.2, HR: 4.2 for patients with MDM2 protein expression) and the logarithmic scale (Score (logrank) test: P<0.0001, CI: 95%, range: 4.5–25.6, HR: 10.7 for patients with MDM2 protein expression) (Figure 1). MDM2-negative MPM showed a 3-year survival of 29%; no patients with MDM2-positive MPM were alive after 3 years.
Figure 1 shows a Kaplan–Meier plot for OS in correlation to the MDM2 immunoexpression in the patient collective from Essen. The x axis shows the survival time in months. On the y axis, the survival rate in percentage is shown. MDM2-positive MPM (regardless of the score) showed a significantly decreased survival time compared with MDM2-negative MPM (P<0.0001).
Analysis of OS with respect to mRNA expression
Out of these 72 samples, 48 specimens were subjected to qPCR analysis. Thirty-eight patients (79%) showed an epithelioid, five (10%) a biphasic and two (4%) a sarcomatoid MPM subtype. For three (6%), the histological subtype remained inconclusive. Five patients (10%) were reported alive, and 43 (90%) had succumbed to the disease. The median OS time was 17.1 months (Table 2).
Sufficient amounts of mRNA for subsequent cDNA synthesis and qPCR analysis could be extracted from all samples. All tested samples returned evaluable qPCR data and were subjected to normalization, and all negative controls (non-template controls) showed no detectable signal.
Neither MDM2 (P=0.54) nor P14/ARF (P=0.27) mRNA expression showed a significant correlation with respect to the patients’ age. P14/ARF mRNA expression showed no significant correlation with respect to sample age (P=0.089). MDM2 mRNA expression showed a statistical trend with respect to sample age (P=0.0505), but the positive rho-value (rho=0.28) indicated a direct correlation between sample age and mRNA leading to the assumption that with increasing age of the sample the mRNA amount would increase. This result was considered as an irrelevant contingency.
OS correlated significantly with MDM2 mRNA expression with respect to the logarithmic scale (Score (logrank) test; P=0.0014) with a hazard ratio of 3.2 for elevated expression (CI: 95%, range: 1.5–6.9). With respect to the linear scale, a significant correlation was found (Score (logrank) test; P=0.0001) confirming the hazard ratio for elevated expression. The results are summarised in Figure 2A.
Statistical analysis of P14/ARF-mRNA expression showed no significant correlation with respect to OS (linear scale: Score (logrank) test; P=0.38, logarithmic scale: Score (logrank) test; P=0.13), but Kaplan–Meier curves separated patients having a low expression from patients with high expression as shown in Figure 2B. Elevated P14/ARF expression correlated with prolonged survival.
The age of the patients (Score (logrank) test; P=0.048) correlated significantly with OS with a hazard ratio of 1.04 (CI: 95%, range: 0.99–1.1) for older patients (data not shown). Gender (Score (logrank) test; P=0.066) showed no significant correlation for OS (data not shown).
Figure 2 shows a Kaplan–Meier plot for OS in correlation to the mRNA expression of (A) MDM2 and (B) P14/ARF. The x axis shows the survival time in months. On the y axis, the survival rate in percentage is shown. Elevated MDM2 expression was associated with significantly decreased survival rates (P<0.0015). P14/ARF expression showed no significant relationship to OS, but Kaplan–Meier curves separated patients with elevated expression from patients with low expression and higher expression correlated with prolonged survival.
Analysis of PFS
All of the investigated patients received cisplatin in combination with pemetrexed. During therapy, 53 (74%) patients showed progression of the disease and 16 (22%) were free of progression. For three patients (4%), no PFS data were available. Median PFS was 6.4 months (mean without censored patients: 9.4 months, range: 4.0–9.3 months) (Table 2). Histological subtype showed a significant correlation with respect to PFS (P<0.0001) with shorter PFS for patients with biphasic and sarcomatoid subtypes (CI: 95%, range: 0.005–0.2, HR: >3.3 for biphasic and sarcomatoid MPM; data not shown). Male patients showed shorter PFS than female patients (P=0.037, CI: 95%, range: 1.0–5.8, HR: 2.4 for male patients; data not shown).
Analysis of PFS with respect to MDM2 immunoexpression
Out of the 65 patients subjected to IHC analysis, 47 (72%) showed progression of the disease, 14 (22%) showed no progression and for 4 (6%) patients no PFS data were available. Median PFS was 6.6 months (Table 2).
Male patients showed a statistically significant shorter PFS than female patients (Score (logrank) test; P=0.037, CI: 95%, range: 1.0–5.8) with a hazard ratio of 2.4 (data not shown). The histological subtype showed a statistically significant correlation to PFS (Score (logrank) test; P<0.0001, CI: 95%, range: 0.2–4.2), with faster progression in biphasic and sarcomatoid MPM (data not shown).
Any MDM2 protein expression (Scores 1–3) correlated with shorter PFS, whereas a lack of MDM2 expression (Score 0) associated with prolonged PFS (linear scale Score (logrank) test; P=0.0004, CI: 95%, range: 1.4–3.7, HR: 2.3 for patients with MDM2 expression and logarithmic scale Score (logrank) test; P=0.0002, CI: 95%, range: 1.99–10.4, HR: 4.5 for patients with MDM2 expression). The results are summarised in Figure 3.
Age of the patients (Score (logrank) test; P=0.75) and age of the specimens (Score (logrank) test; P=0.51) showed no statistical correlation to PFS.
Figure 3 shows a Kaplan–Meier plot for PFS in correlation to the protein expression of MDM2. On the x axis, the survival time in months is shown. The y axis shows the survival rate in percentage. Higher MDM2 expression (regardless of the score) was significantly associated with shorter PFS (P<0.0005).
Analysis of PFS with respect to mRNA expression
Out of the 48 patients who were investigated by qPCR, 38 (73%) showed progression of the disease, whereas 11 (23%) were free of progression and for 3 (6%) no PFS data were available. Median PFS was 6.1 months (Table 2). For PFS, a significant correlation for MDM2-mRNA expression was found with respect to the logarithmic scale (Score (logrank) test; P=0.0072, CI: 95%, range 1.3–5.4) and also with respect to the linear scale (Score (logrank) test; P=0.0049) as shown in Figure 4A. Elevated expression showed a hazard ratio of 2.6 with shorter PFS in patients with elevated MDM2 expression.
Statistical analysis of P14/ARF mRNA expression showed a statistical trend with elevated expression in patients with prolonged PFS (logarithmic scale: Score (logrank) test; P=0.057 and linear scale: Score (logrank) test; P=0.62). Figure 4B shows the Kaplan–Meier curves for P14/ARF expression with respect to PFS.
The patients’ age (Score (logrank) test; P=0.63) and age of the specimens (Score (logrank) test; P=0.15) did not show any impact on PFS (data not shown).
Figure 4 shows a Kaplan–Meier plot for PFS in correlation to the mRNA expression of (A) MDM2 and (B) P14/ARF. On the x axis, the survival time in months is shown. The y axis shows the survival rate in percentage. Higher MDM2 expression was significantly associated with shorter PFS (P<0.0015). P14/ARF expression showed a statistical trend correlating prolonged PFS with elevated expression (P=0.057).
Discussion
The standard first-line therapy for MPM is a combination of cisplatin or carboplatin with pemetrexed (Ramalingam and Belani, 2008; Ray and Kindler, 2009), and this treatment results in a median OS of 11–12 months (Papa et al, 2013). Our investigated patients showed a comparable median OS of 18.5 months. The investigated patients showed a median PFS of 6.4 months that is comparable to previously reported PFS of 5.7 months (Ramalingam and Belani, 2008; Ray and Kindler, 2009). The efficacy of platin-based therapies depends on several DNA-repair enzymes, which determine the potential of a neoplasia to respond to platin-induced damage (Ting et al, 2013). These rather short OS and PFS rates imply that the standard MPM therapy can be considered deficient (Tomek and Manegold, 2004; Guyatt et al, 2006; Muers et al, 2008; Stahel et al, 2009; Astoul et al, 2012), and predictive biomarkers for cisplatin–pemetrexed-based therapy concepts are lacking or discussed controversially (Tomek and Manegold, 2004; Stahel et al, 2009; Astoul et al, 2012; Mairinger et al, 2013a). Additionally, no approved second-line therapy exists (Papa et al, 2013). Therefore, a recent guideline emphasises the need of an innovative and novel therapy strategy (Astoul et al, 2012) that should be based on reliable, predictive and prognostic biomarkers.
The tumour-suppressor TP53 gene locus is mutated in approximately 50% of all human cancers. In the remaining 50% of all malignant tumours, TP53 is wild type but inactivated. TP53 mutations are extremely rare events in MPM, but the P53 protein can be inactivated by several other molecular mechanisms (Papp et al, 2001a, 2001b; Toyooka et al, 2008). One of these putative mechanisms is an amplification and/or overexpression of MDM2, which exerts an E3 ubiquitin protein ligase function and thus is a physiological repressor of the P53 functional protein (Jones et al, 1995; Montes de Oca Luna et al, 1995; Parant et al, 2001; Marine et al, 2006; Ringshausen et al, 2006). Furthermore, MDM2 expression is regulated by transcriptionally active P53 showing that both MDM2 and P53 control each other in a (negative) feedback loop manner (Wang et al, 2011; Rao et al, 2013). This is an important physiological interplay, keeping P53 levels low under non-cancerous, physiological cell conditions and inducing P53 activity in the presence of cellular stress (Hopkins-Donaldson et al, 2006; Herce et al, 2013). Overexpression of MDM2 can lead to a loss of P53 regulatory function in cancer cells (Jones et al, 1995; Montes de Oca Luna et al, 1995; Parant et al, 2001; Marine et al, 2006; Ringshausen et al, 2006). Of note, enzymes involved in DNA repair, such as P53, are associated with patients’ response and outcome to platinum-based therapy regimens (Barckhausen et al, 2014; Sun et al, 2014), and P53’s activity and stability is negatively regulated by overexpression of MDM2 in approximately 20% of epithelioid mesotheliomas and the epithelioid component of biphasic MPM (Mairinger et al, 2014). In the recent manuscript, we confirmed this previous finding and additionally found that MDM2 has prognostic value on the mRNA and protein level. Prolonged survival and PFS in MPM patients was associated with lower expression of MDM2 on the mRNA level and absent MDM2 immunoexpression. Furthermore, MDM2-negative MPM showed a 3-year survival of 29%; no patients with MDM2-positive MPM were alive after 3 years. This is in line with previous findings (Mairinger et al, 2014). Expression analysis on the protein and mRNA level showed that the statistical results were highly consistent between both methods indicating that IHC and qPCR could be used interchangeably to test MPM patients for the expression of MDM2. FFPE tissue is a challenging source for molecular biological analysis, but if the assay design takes potential FFPE-specific pitfalls into account, reliable and reproducible results can be achieved (Walter et al, 2013).
Another important tumour suppressor in this setting is P14/ARF that stabilises P53 (Rao et al, 2013). P14/ARF is the physiological inhibitor of MDM2 and, when bound to MDM2, prevents P53 degradation (Rao et al, 2013). Additionally, P14/ARF can increase P53 synthesis, inhibit other negative regulators of P53 than MDM2, regulate P53 influencing pathways, increase the transcriptional activity of P53 and has a critical role as tumour suppressor (Huang et al, 2003; Chen et al, 2005; Rocha et al, 2005; Miao et al, 2010). Loss of P14/ARF is expected to have a similar impact on cancer development and maintenance as inactivation of TP53 (Kanellou et al, 2009). P14/ARF is encoded from the CDKN2A gene locus that also encodes P16/INK2A (Chen et al, 2005). P16/INK2A and P14/ARF are controlled by distinct promoters and differ in one exon leading to two unique proteins that are structurally different (Kanellou et al, 2009). Deletion of the whole CDKN2A locus, on chromosome 9p21, is present in 79–90% of all malignant mesotheliomas; in the sarcomatoid subtype, the prevalence is up to 100% (Frew et al, 2009; Krasinskas et al, 2010; Altomare et al, 2011; Monaco et al, 2011; Bahnassy et al, 2012; Matsumoto et al, 2013; Tochigi et al, 2013). The inactivation of the CDKN2A locus occurs also without deletion. Epigenetic inactivation including DNA methylation (Kanellou et al, 2009; Fujii et al, 2012) and miRNA regulation were reported (Guled et al, 2009; Ivanov et al, 2010). In this study, P14/ARF expression reached no statistical significance, but Kaplan–Meier curves separated patients with low expression and poor prognosis from patients with high expression and favourable prognosis. Furthermore, P14/ARF showed a statistical trend with respect to PFS, and low expression was associated with faster progression of the disease. In an in vitro experiment, P14/ARF was identified as a marker for response to Nutlin-3A treatment, which is a potent and selective MDM2 inhibitor (Van Maerken et al, 2011). This substantiates the notion that MDM2-mediated P53 inactivation may benefit from inactivation of P14/ARF.
Nutlin-3A (a cis-imidazoline analogue) is a newly developed, potent and selective MDM2 inhibitor with an IC50 value in the 90–300 nM range (Vassilev et al, 2004; Shangary and Wang, 2009) that prevents MDM2–P53 interaction by binding to the hydrophobic P53-binding pocket of MDM2 (Vassilev et al, 2004; Gamble et al, 2012). It is a non-genotoxic drug that can restore P53 activity leading to subsequent senescence or apoptosis in a cell-type-dependent manner (Vassilev et al, 2004; Gamble et al, 2012; Voltan et al, 2013). Additionally, Nutlin-3A shows a low risk of inducing therapy resistance and is currently being tested in a phase I clinical trial (protocol ID: NCT00623870 as substance RO5045337) (Voltan et al, 2013). In summary, MDM2 overexpression seems to mediate an inactivation of P53 leading to more aggressive MPM that are less sensitive to current therapies resulting in poor outcome, and additional downregulation of its physiological inhibitor P14/ARF may intensify this effect. In a previous study, P53 failed as prognostic factor in MPM patients (Mairinger et al, 2014), but the assessment of MDM2 may be a helpful tool in a subset of MPM patients to identify patients who would have the largest benefit from a platin–pemetrexed therapy and to predict the outcome.
Conclusion
Despite being wild type with respect to TP53, P53-mediated cell cycle control or apoptosis are lacking in MPM, which might be explained by inactivation of functional P53 protein by different mechanisms, such as proteasomal degradation via overexpression of MDM2. Overall survival and PFS showed a significant correlation between increased MDM2 expression and decreased survival, making MDM2 a reliable and robust prognostic and predictive biomarker in MPM.
P14/ARF mRNA expression is significantly decreased in many MPM, which might further contribute to deregulation and hyperfunction of MDM2. Kaplan–Meier curves were able to separate patients with low P14/ARF expression with poor outcome from patients with higher expression and favourable outcome.
In summary, a substantial proportion of MPM show a MDM2-mediated inactivation of P53 and concomitant downregulation of P14/ARF that can predict the response to pemetrexed and platinum-based chemotherapy regimens.
Change history
03 March 2015
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Altomare DA, Menges CW, Xu J, Pei J, Zhang L, Tadevosyan A, Neumann-Domer E, Liu Z, Carbone M, Chudoba I, Klein-Szanto AJ, Testa JR (2011) Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis. PLoS One 6: e18828.
Astoul P, Roca E, Galateau-Salle F, Scherpereel A (2012) Malignant pleural mesothelioma: from the bench to the bedside. Respiration 83: 481–493.
Bahnassy AA, Zekri AR, Abou-Bakr AA, El-Deftar MM, El-Bastawisy A, Sakr MA, El-Sherif GM, Gaafar RM (2012) Aberrant expression of cell cycle regulatory genes predicts overall and disease free survival in malignant pleural mesothelioma patients. Exp Mol Pathol 93: 154–161.
Barckhausen C, Roos WP, Naumann SC, Kaina B (2014) Malignant melanoma cells acquire resistance to DNA interstrand cross-linking chemotherapeutics by p53-triggered upregulation of DDB2/XPC-mediated DNA repair. Oncogene 33: 1964–1974.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622.
Chen D, Kon N, Li M, Zhang W, Qin J, Gu W (2005) ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 121: 1071–1083.
Christoph DC, Asuncion BR, Mascaux C, Tran C, Lu X, Wynes MW, Gauler TC, Wohlschlaeger J, Theegarten D, Neumann V, Hepp R, Welter S, Stamatis G, Tannapfel A, Schuler M, Eberhardt WE, Hirsch FR (2012) Folylpoly-glutamate synthetase expression is associated with tumor response and outcome from pemetrexed-based chemotherapy in malignant pleural mesothelioma. J Thorac Oncol 7: 1440–1448.
Flores RM, Zakowski M, Venkatraman E, Krug L, Rosenzweig K, Dycoco J, Lee C, Yeoh C, Bains M, Rusch V (2007) Prognostic factors in the treatment of malignant pleural mesothelioma at a large tertiary referral center. J Thorac Oncol 2: 957–965.
Frew AJ, Johnstone RW, Bolden JE (2009) Enhancing the apoptotic and therapeutic effects of HDAC inhibitors. Cancer Lett 280: 125–133.
Fujii M, Fujimoto N, Hiraki A, Gemba K, Aoe K, Umemura S, Katayama H, Takigawa N, Kiura K, Tanimoto M, Kishimoto T (2012) Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma. Cancer Sci 103: 510–514.
Gamble LD, Kees UR, Tweddle DA, Lunec J (2012) MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63. Oncogene 31: 752–763.
Goudar RK (2008) Review of pemetrexed in combination with cisplatin for the treatment of malignant pleural mesothelioma. Ther Clin Risk Manag 4: 205–211.
Guled M, Lahti L, Lindholm PM, Salmenkivi K, Bagwan I, Nicholson AG, Knuutila S (2009) CDKN2A, NF2, and JUN are dysregulated among other genes by miRNAs in malignant mesothelioma -a miRNA microarray analysis. Genes Chromosomes Cancer 48: 615–623.
Guyatt G, Gutterman D, Baumann MH, Addrizzo-Harris D, Hylek EM, Phillips B, Raskob G, Lewis SZ, Schunemann H (2006) Grading strength of recommendations and quality of evidence in clinical guidelines: report from an american college of chest physicians task force. Chest 129: 174–181.
Harris SL, Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24: 2899–2908.
Hazarika M, White RM Jr, Booth BP, Wang YC, Ham DY, Liang CY, Rahman A, Gobburu JV, Li N, Sridhara R, Morse DE, Lostritto R, Garvey P, Johnson JR, Pazdur R (2005) Pemetrexed in malignant pleural mesothelioma. Clin Cancer Res 11: 982–992.
Herce HD, Deng W, Helma J, Leonhardt H, Cardoso MC (2013) Visualization and targeted disruption of protein interactions in living cells. Nat Commun 4: 2660.
Hopkins-Donaldson S, Belyanskaya LL, Simoes-Wust AP, Sigrist B, Kurtz S, Zangemeister-Wittke U, Stahel R (2006) p53-induced apoptosis occurs in the absence of p14(ARF) in malignant pleural mesothelioma. Neoplasia 8: 551–559.
Huang Y, Tyler T, Saadatmandi N, Lee C, Borgstrom P, Gjerset RA (2003) Enhanced tumor suppression by a p14ARF/p53 bicistronic adenovirus through increased p53 protein translation and stability. Cancer Res 63: 3646–3653.
Ivanov SV, Goparaju CM, Lopez P, Zavadil J, Toren-Haritan G, Rosenwald S, Hoshen M, Chajut A, Cohen D, Pass HI (2010) Pro-tumorigenic effects of miR-31 loss in mesothelioma. J Biol Chem 285: 22809–22817.
Jones SN, Roe AE, Donehower LA, Bradley A (1995) Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378: 206–208.
Kanellou P, Zaravinos A, Zioga M, Spandidos DA (2009) Deregulation of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in basal cell carcinoma. Br J Dermatol 160: 1215–1221.
Kindler HL (2008) Systemic treatments for mesothelioma: standard and novel. Curr Treat Options Oncol 9: 171–179.
Krasinskas AM, Bartlett DL, Cieply K, Dacic S (2010) CDKN2A and MTAP deletions in peritoneal mesotheliomas are correlated with loss of p16 protein expression and poor survival. Mod Pathol 23: 531–538.
Lustgarten DE, Deshpande C, Aggarwal C, Wang LC, Saloura V, Vachani A, Wang LP, Litzky L, Feldman M, Creaney J, Nowak AK, Langer C, Inghilleri S, Stella G, Albelda SM (2013) Thymidylate synthase and folyl-polyglutamate synthase are not clinically useful markers of response to pemetrexed in patients with malignant pleural mesothelioma. J Thorac Oncol 8: 469–477.
Mairinger F, Vollbrecht C, Halbwedl I, Hatz M, Stacher E, Gully C, Quehenberger F, Stephan-Falkenau S, Kollmeier J, Roth A, Mairinger T, Popper H (2013a) Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma. J Thorac Oncol 8: 644–653.
Mairinger F, Vollbrecht C, Mairinger T, Popper H (2013b) The issue of studies evaluating biomarkers which predict outcome after pemetrexed-based chemotherapy in malignant pleural mesothelioma. J Thorac Oncol 8: e80–e82.
Mairinger FD, Walter RF, Ting S, Vollbrecht C, Kollmeier J, Griff S, Hager T, Mairinger T, Christoph DC, Theegarten D, Kurt Werner S, Wohlschlaeger J (2014) Mdm2 protein expression is strongly associated with survival in malignant pleural mesothelioma. Future Oncol 10: 995–1005.
Marine JC, Francoz S, Maetens M, Wahl G, Toledo F, Lozano G (2006) Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ 13: 927–934.
Matsumoto S, Nabeshima K, Kamei T, Hiroshima K, Kawahara K, Hata S, Marukawa K, Matsuno Y, Taguchi K, Tsujimura T (2013) Morphology of 9p21 homozygous deletion-positive pleural mesothelioma cells analyzed using fluorescence in situ hybridization and virtual microscope system in effusion cytology. Cancer Cytopathol 121: 415–422.
Miao L, Song Z, Jin L, Zhu YM, Wen LP, Wu M (2010) ARF antagonizes the ability of Miz-1 to inhibit p53-mediated transactivation. Oncogene 29: 711–722.
Monaco SE, Shuai Y, Bansal M, Krasinskas AM, Dacic S (2011) The diagnostic utility of p16 FISH and GLUT-1 immunohistochemical analysis in mesothelial proliferations. Am J Clin Pathol 135: 619–627.
Montes de Oca Luna R, Wagner DS, Lozano G (1995) Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378: 203–206.
Muers MF, Stephens RJ, Fisher P, Darlison L, Higgs CM, Lowry E, Nicholson AG, O'brien M, Peake M, Rudd R, Snee M, Steele J, Girling DJ, Nankivell M, Pugh C, Parmar MK Group, M. S. T. M. (2008) Active symptom control with or without chemotherapy in the treatment of patients with malignant pleural mesothelioma (MS01): a multicentre randomised trial. Lancet 371: 1685–1694.
Papa S, Popat S, Shah R, Prevost AT, Lal R, Mclennan B, Cane P, Lang-Lazdunski L, Viney Z, Dunn JT, Barrington S, Landau D, Spicer J (2013) Phase 2 study of sorafenib in malignant mesothelioma previously treated with platinum-containing chemotherapy. J Thorac Oncol 8: 783–787.
Papp T, Schipper H, Pemsel H, Bastrop R, Muller KM, Wiethege T, Weiss DG, Dopp E, Schiffmann D, Rahman Q (2001a) Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas. Int J Oncol 18: 425–433.
Papp T, Schipper H, Pemsel H, Unverricht M, Muller KM, Wiethege T, Schiffmann D, Rahman Q (2001b) Mutational analysis of the PTEN/MMAC1 tumour suppressor gene in primary human malignant mesotheliomas. Oncol Rep 8: 1375–1379.
Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke V, Jochemsen AG, Lozano G (2001) Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet 29: 92–95.
Ramalingam SS, Belani CP (2008) Recent advances in the treatment of malignant pleural mesothelioma. J Thorac Oncol 3: 1056–1064.
Rao B, Lain S, Thompson AM (2013) p53-Based cyclotherapy: exploiting the ‘guardian of the genome' to protect normal cells from cytotoxic therapy. Br J Cancer 109: 2954–2958.
Ray M, Kindler HL (2009) Malignant pleural mesothelioma: an update on biomarkers and treatment. Chest 136: 888–896.
Righi L, Papotti MG, Ceppi P, Bille A, Bacillo E, Molinaro L, Ruffini E, Scagliotti GV, Selvaggi G (2010) Thymidylate synthase but not excision repair cross-complementation group 1 tumor expression predicts outcome in patients with malignant pleural mesothelioma treated with pemetrexed-based chemotherapy. J Clin Oncol 28: 1534–1539.
Ringshausen I, O'shea CC, Finch AJ, Swigart LB, Evan GI (2006) Mdm2 is critically and continuously required to suppress lethal p53 activity in vivo. Cancer Cell 10: 501–514.
Rocha S, Garrett MD, Campbell KJ, Schumm K, Perkins ND (2005) Regulation of NF-kappaB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor. EMBO J 24: 1157–1169.
Rusch VW, Giroux D, Kennedy C, Ruffini E, Cangir AK, Rice D, Pass H, Asamura H, Waller D, Edwards J, Weder W, Hoffmann H, Van Meerbeeck JP Committee, IS (2012) Initial analysis of the international association for the study of lung cancer mesothelioma database. J Thorac Oncol 7: 1631–1639.
Shangary S, Wang S (2009) Small-molecule inhibitors of the MDM2-p53 protein-protein interaction to reactivate p53 function: a novel approach for cancer therapy. Annu Rev Pharmacol Toxicol 49: 223–241.
Stahel RA, Weder W, Felip E Group, E. G. W. (2009) Malignant pleural mesothelioma: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol 20 (Suppl 4): 73–75.
Sun H, Wang Y, Wang Z, Meng J, Qi Z, Yang G (2014) Aurora-A controls cancer cell radio- and chemoresistance via ATM/Chk2-mediated DNA repair networks. Biochim Biophys Acta 1843: 934–944.
Ting S, Mairinger FD, Hager T, Welter S, Eberhardt WE, Wohlschlaeger J, Schmid KW, Christoph DC (2013) ERCC1, MLH1, MSH2, MSH6, and betaIII-tubulin: resistance proteins associated with response and outcome to platinum-based chemotherapy in malignant pleural mesothelioma. Clin Lung Cancer 14: 558–567, e3.
Tochigi N, Attanoos R, Chirieac LR, Allen TC, Cagle PT, Dacic S (2013) p16 Deletion in sarcomatoid tumors of the lung and pleura. Arch Pathol Lab Med 137: 632–636.
Toledo F, Wahl GM (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6: 909–923.
Tomek S, Manegold C (2004) Chemotherapy for malignant pleural mesothelioma: past results and recent developments. Lung Cancer 45 (Suppl 1): S103–S119.
Toyooka S, Kishimoto T, Date H (2008) Advances in the molecular biology of malignant mesothelioma. Acta Med Okayama 62: 1–7.
Treasure T, Sedrakyan A (2004) Pleural mesothelioma: little evidence, still time to do trials. Lancet 364: 1183–1185.
Tsao AS, Wistuba I, Roth JA, Kindler HL (2009) Malignant pleural mesothelioma. J Clin Oncol 27: 2081–2090.
Van Maerken T, Rihani A, Dreidax D, De Clercq S, Yigit N, Marine JC, Westermann F, De Paepe A, Vandesompele J, Speleman F (2011) Functional analysis of the p53 pathway in neuroblastoma cells using the small-molecule MDM2 antagonist nutlin-3. Mol Cancer Ther 10: 983–993.
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303: 844–848.
Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, Gatzemeier U, Boyer M, Emri S, Manegold C, Niyikiza C, Paoletti P (2003) Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21: 2636–2644.
Voltan R, Secchiero P, Ruozi B, Forni F, Agostinis C, Caruso L, Vandelli MA, Zauli G (2013) Nanoparticles engineered with rituximab and loaded with Nutlin-3 show promising therapeutic activity in B-leukemic xenografts. Clin Cancer Res 19: 3871–3880.
Walter RF, Mairinger FD, Wohlschlaeger J, Worm K, Ting S, Vollbrecht C, Kurt Werner S, Hager T (2013) FFPE tissue as a feasible source for gene expression analysis—a comparison of three reference genes and one tumor marker. Pathol Res Pract 209: 784–789.
Wang J, Zheng T, Chen X, Song X, Meng X, Bhatta N, Pan S, Jiang H, Liu L (2011) MDM2 antagonist can inhibit tumor growth in hepatocellular carcinoma with different types of p53 in vitro. J Gastroenterol Hepatol 26: 371–377.
Weill H, Hughes JM, Churg AM (2004) Changing trends in US mesothelioma incidence. Occup Environ Med 61: 438–441.
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Walter, R., Mairinger, F., Ting, S. et al. MDM2 is an important prognostic and predictive factor for platin–pemetrexed therapy in malignant pleural mesotheliomas and deregulation of P14/ARF (encoded by CDKN2A) seems to contribute to an MDM2-driven inactivation of P53. Br J Cancer 112, 883–890 (2015). https://doi.org/10.1038/bjc.2015.27
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DOI: https://doi.org/10.1038/bjc.2015.27
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