| | Combined effects of MDM2 SNP 309 and p53 mutation on oral squamous cell carcinomas associated with areca quid chewingReceived 5 February 2008; accepted 12 March 2008. published online 09 June 2008. Introduction  The tumor suppressor gene p53 is one of the most commonly mutated genes in human cancer.1, 2 It functions primarily as a transcription factor and can mediate its different downstream functions by activating or repressing a large number of target genes including MDM2.3, 4, 5, 6 p53 and MDM2 form an autoregulatory loop in which the intracellular p53 level was regulated. p53 induces the transcription of MDM2 which encodes a ubiquitin protein ligase that regulates the stability of p53 by targeting it for proteosomal degradation. Overexpression of MDM2 can yield a phenotype similar to that of the mutational inactivation of p53.7 The single nucleotide polymorphism (SNP), T309G, identified in the MDM2 promoter region has been shown to increase the binding affinity of the transcriptional activator Sp1 to the MDM2 promoter, resulting in a higher expression of MDM2 mRNA and protein and subsequent attenuation of the p53 pathway.8 Bond et al.8 studied a cohort of 88 patients with Li–Fraumeni syndrome, a germline mutation in one p53 allele, and found that the MDM2 SNP 309 GG genotype was associated with early tumor onset and multiple primary cancers. The p53 gene is one of the most highly mutated genes in human tumors, and tumors with p53 mutations have been found to associate with a more aggressive phenotype and a worse prognosis in multiple tumor types.9 Because somatic p53 gene mutation is such a frequent event in tumors and associates with altered cancer progression, a complete understanding of the effects of functional germline variations in the p53 pathway can only be gained by incorporating the mutational status of the p53 gene into studies. Only a few recently studies describe the interactions between MDM2 SNP 309 and the somatic p53 mutational status in both cancer incidence10 and survival11 and none in head and neck cancer or oral squamous cell carcinoma (OSCC). Since the cancer acceleration associated with MDM2 SNP 309 was more frequent in the younger woman which may be related to their typically higher hormone levels, particularly estrogen and suggest that an active estrogen-signaling pathway is needed for the G allele to accelerate tumor formation in humans.8, 12 OSCC in Taiwan occurred mostly in male and was associated with the use of areca quid (AQ) chewing, cigarette smoking and alcohol drinking.13 Areca nut seed contains diosgenin and beta-sitosterol which have been known for possessing estrogenic activities.14 Furthermore, AQ chewing could transiently increase estrogen secretion in saliva.15 Therefore, the effects of MDM2 SNP 309 on the cancer development in men with habitual AQ chewing would be highly conceivable. In this study, we investigated the combined effects of MDM2 SNP 309 and p53 mutations on the clinical outcomes of Taiwanese OSCC patients. Patients and methods Patients, tissue specimens and clinical diagnosis This study was approved by the Institutional Review Board, Chang Gung Memorial Hospital. A total of 480 OSCC patients treated at Chang Gung Memorial Hospital, Lin-Kuo, during the period from August 1996 to November 2001 were recruited to participate in the study. All the cases were histologically confirmed. Female patients (n =30) and males diagnosed with nonsquamous cell carcinoma (n=30) were excluded from this study due to insufficient numbers. Tumors located at the hypopharynx and oropharynx (n=59) were also excluded because their contributive risk factors and clinical behaviors might be different from oral cavity tumors. Patients without the status of lymph node extracapsular spread (LNECS) (n=10) were also excluded. Thus, 351 male oral cavity OSCC patients, including 237 patients previously studied,16 were included in the present analysis. All the patients gave informed consent for participation and were interviewed uniformly before surgery by a well-trained interviewer. The questionnaire used in the interview sought detailed information on current and past cigarette smoking, alcohol drinking, AQ chewing habits, family disease history, and general demographic data. For each case, a tumor sample was surgically dissected into small pieces, frozen immediately in liquid nitrogen and stored at −80°C. In addition, 10ml of venous blood was drawn, separated into plasma, buffy coat and red blood cells by centrifugation within 18h of obtaining the blood, and stored at −80°C. Genomic DNA for genotyping was purified from the buffy coat as described.16 As controls, 1272 males with available blood samples were included in this study and were selected from 3000 random samples of the Taiwanese general population originally collected to study blood lead concentrations.17 MDM2 SNP 309 genotyping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) The MDM2 SNP 309 was determined according to a MALDI-TOF method as described.18, 19 The primer sets used for PCR amplification of the MDM2 SNP 309 region are as described by Bond et al.8 Briefly, 0.1μg genomic DNA was amplified by PCR in a final volume of 25μL following the protocol described by Bond et al.8 for 30 cycles (1min at 95°C, 1min at 62°C, and 1min at 72°C) using a Mastercycler® gradient (Eppendorf AG, Hamburg, Germany). The unincorporated dNTPs and primers were removed automatically via MAPIIA (GenePure PCR purification system, Bruker, Bremen, Germany). The purified PCR products were collected, and primer extension reactions were conducted using the downstream MDM2 SNP 309 primer (5′-CCG GAC CTC CCG CGC CG-3′) [rs2279744] in a 20-μL solution containing 50ng PCR product, 10pmol primer, 0.5μL of 1mM ddCTP/dNTP, 0.5U of Thermo Sequenase DNA polymerase (Amersham Biosciences, Piscataway, NJ), and 2μL reaction buffer provided by the manufacturer. The reaction was carried out in a multiblock system thermocycler (ThermoHybaid, Waltham, MA) with an initial denaturation step at 96°C for 75s followed by 49 cycles (96°C for 15s, 43°C for 15s, 60°C for 100s), then 96°C for 30s. The primer extension reaction products were purified automatically via MAPIIA (single-strand DNA binding beads, Bruker) and analyzed by MALTI-TOF MS. Prior to MALDI-TOF MS analysis, the sample was mixed with 0.5μL of matrix solution (50mg/ml 3-hydropicolinic acid in a 4:5:1 mixture of water, acetonitrile and 50mg/ml diammonium citrate) and spotted on a 384-well Teflon sample plate (PerSeptive Biosystems, Framingham, MA). MALDI-TOF mass spectra were acquired using a Bruker Autoflex MALDI-TOF and AutoXecute software (Bruker) as validating tools for genotyping data. Mutation analysis of the p53 gene Single-stranded conformation polymorphism (SSCP) analysis was used to analyze tumor samples for mutations within exons 4–10 of the p53 gene as described.20, 21 Cases displaying an altered electrophoretic mobility were reamplified and analyzed by direct sequencing of both strands to confirm and characterize the nature of the mutation. Statistical analysis Statistical analyses were performed with the SPSS statistical package version 8.0 (SPSS, Chicago, IL). The differences in age of OSCC onset between MDM2 SNP 309 genotypes were examined by one-way ANOVA. The associations between the frequency of MDM2 SNP 309, p53 mutation and clinicopathologic factors including tumor cell differentiation, tumor staging (according to 1997 American Joint Committee on Cancer staging criteria), lymph node extracapsular spread (LNECS), cigarette smoking, alcohol drinking, and AQ chewing were examined with the χ2 test or Fisher’s exact test. Survival curves were constructed with the Kaplan–Meier method and compared with the log-rank test. The Cox regression model was applied to estimate the hazard ratio (HR) and 95% confidence interval (CI) for the MDM2 SNP 309 and p53 mutation. A two-sided value of p<0.05 was considered statistically significant. Results A total of 351 OSCC patients were enrolled in this study. Table 1 presents the demographic information and clinicopathological characteristics of the patients. The median follow-up for all the patients was 28.2 months. The MDM2 SNP 309 G allele frequency was 0.530 and 0.521 among the 1272 controls and 351 OSCC patients, respectively. The observed genotype frequencies of the MDM2 SNP 309 in controls did not deviate significantly from those expected from the Hardy–Weinberg equilibrium (p=0.31) and were not different among three major ethnic groups (Fukien-Taiwanese, Hakkanese and Mainlander) in Taiwan. The frequencies of MDM2 SNP 309 TT, TG, and GG genotypes among OSCC patients were not different from those among controls (p=0.87). There was no correlation between MDM2 SNP 309 and alcohol drinking, cigarette smoking, or AQ chewing (Table 2). The mean age was 51.76 (SD=11.76, n=80), 48.85 (SD=9.91, n=176), and 47.31 (SD=11.26, n=95) years for OSCC patients with the MDM2 SNP 309 TT, TG and GG genotype, respectively. Table 3 indicates that the G allele was associated with poor differentiation of the tumor (χ2 trend test, p=0.01). Among 351 OSCC patients, tumor samples from 273 patients without antecedent treatment, including 237 cases published previously,20 were examined for p53 gene mutations by PCR-SSCP and direct sequencing. As shown in Table 3, no significant correlation was found between the MDM2 SNP 309 genotype and the frequency of p53 gene mutation (p=0.86), lymph node metastasis (p=0.72) or LNECS (p=0.72). However, the frequency of LNECS was increased in MDM2 SNP 309 GG individuals with p53 mutation (χ2 trend test, p=0.04, Table 4). | ⁎ p<0.05. |
p53 mutations were not associated with disease-free survival (DFS) in this series of OSCC patients by univariate analysis (p=0.94, Fig. 1A). However, the combination of MDM2 SNP 309 GG genotype with p53 mutation was associated with poor DFS in early stage OSCC patients (log-rank test p=0.05; HR=2.77, Fig. 1B). For advanced stage with lymph node positive OSCC patients, the combination of MDM2 SNP 309 GG genotype with p53 mutation was associated with poor DFS (log-rank test p=0.04; HR=1.93, Fig. 1C); while the combination of MDM2 SNP 309 GG genotype with p53 mutation was not associated with poor DFS in lymph node negative advanced stage OSCC patients (log-rank test p=0.95; HR=0.97, data not shown). Discussion p53 controls several key pathways that protect cells from malignant transformation. Approximately 40–50% of OSCC have alterations in p53,20 which regulates the cellular stress response pathway that has been shown to be critical for the maintenance of genomic integrity. Genes such as MDM2 that interfere with the p53-cellular stress response pathway might function as modifiers of individual cancer risks or the tumor behaviors.22 Recently, Bond et al.8 reported the association between SNP 309 in the MDM2 promoter and MDM2 transcriptional levels and showed that Li–Fraumani syndrome patients with the MDM2 SNP 309 GG genotype developed soft tissue sarcoma 12 years earlier, on average, than those with the MDM2 SNP 309 TT genotype. In our study, the mean age of onset for male OSCC patients with the MDM2 SNP 309 GG genotype was 4.45 years earlier than those with the MDM2 SNP 309 TT genotype (p=0.02). The patients harboring GG genotype could have attenuated p53 pathway and impaired genomic repair ability. It has been shown that the MDM2-transgenic mice developed spontaneous tumors throughout their lifetime.23 It was also reported that the targeted expression of MDM2 in the murine epidermis increased papilloma formation induced by chemical carcinogen and predisposed to the appearance of premalignant lesions and squamous cell carcinoma.24 Individuals carrying MDM2 SNP 309 GG genotype have their oral mucosa more susceptible to carcinogen exposure (cigarette, alcohol and AQ) and resulted in the earlier onset of tumor formation. Genomic changes such as p53 mutations occurred early and were critical in the tumorigenesis of OSCC.20 However, MDM2 SNP 309 GG genotype did not significantly increase patients’ susceptibility to p53 mutations. Thus, this functional SNP is not only associated with earlier age of the onset of hereditary cancers but also with earlier development of sporadic cancers such as OSCC in Taiwanese males. In the present study, male OSCC patients with the MDM2 SNP 309 GG genotype had a significantly higher frequency of poorly differentiated tumors than those with T alleles (p=0.01). Recently, Hong et al.25 also found that patients carrying the MDM2 SNP 309 GG genotype had an increased risk of developing poorly differentiated and advanced esophageal squamous cell carcinoma at the time of diagnosis. Dazard et al.26 demonstrated an important role for p53 in ensuring correct coordination of cell cycle and cell division with genome integrity in epidermal basal keratinocytes. In contrast, high levels of MDM2 might contribute to sustaining active cell cycle and cellular growth as these cells lose their proliferative potential and initiate terminal differentiation. Thus, the data to date indicate that the p53/MDM2 pathway might play a critical role in the regulation of the epidermal transition from proliferation to differentiation. LNECS is described as an extranodal extension of tumors outside the lymph node capsule. In head and neck tumors, LNECS was shown to be an important factor adversely affecting survival.27, 28 In this series of OSCC patients, we also found LNECS was associated with poor DFS (HR=2.08; 95% CI, 1.15–3.76). Co-expression of MDM2 and p53 was reported to be associated with lymph node metastasis in oral cancers and may also be associated with aggressive tumor behavior.29 In this study, tumors with p53 mutations from male OSCC patients carrying the MDM2 SNP 309 GG genotype had a higher frequency of LNECS at the time of diagnosis (p=0.04 by χ2 trend test). As expected, the combination of MDM2 SNP 309 GG genotype with p53 mutation was associated with poor DFS in lymph node positive advanced stage OSCC patients (HR=1.93; 95% CI, 1.00–3.73). Our results indicate an interaction between MDM2 SNP 309 and p53 with respect to tumor behavior. Accumulating evidence shows that the overexpression of MDM2 is associated with increased tumor metastasis.30 With regard to OSCC, it has been shown that the MDM2 and p53 overexpression was significantly related to neck lymph node metastasis.29 The mechanism behind the increase in metastasis could be related to the stress response in p53/MDM2 pathway. Hypoxia up-regulates MDM2 in a p53-independent manner. The up-regulated MDM2 is associated with the inhibition of p53 downstream pro-apoptotic genes and leads to an increased metastatic efficiency in cell lines.31 In addition, the elevated expression of p53 and MDM-2 protein correlated with increased vascular endothelial growth factor expression,32 which can lead to the growth of metastases and facilitate extravasation of tumor cells.33 When MDM2 overexpression was considered together with p53 inactivation, it was significantly correlated with the degradation of the basement membrane in superficial urothelial carcinoma,34 allowing the cancer to spread. Thus, p53 mutation acting synergistically with MDM2 overexpression increases the risk of metastasis, and extracapsular spread of metastatic foci. This is consistent with the notion that MDM2 regulates the p53 pathway35 and with the premise that two risk factors acting in the same causal pathway result in multiplicative effects on the aggressiveness of tumor cells.36 The prognostic implications of p53 mutations37, 38 and MDM2 expression in OSCCs remain controversial.39, 40 In the present study, p53 mutation alone was not affecting the DFS in OSCC patients. The combination of GG genotype with p53 mutation was associated with poor DFS in early stage OSCC patients (Fig. 1B). This finding is consistent with the previously reported function of mutant p53 and SNP 309.8, 41 The mutant form of p53 has a dominant gain-of-function activity or it may block the wild-type protein by acting as a dominant negative.42 MDM2 further regulates wild-type p53 stability by shuttling p53 from the nucleus to the cytoplasm where it mediates p53 degradation and promotes the degradation of wild-type p53 by the ubiquitin-proteasome system. In addition, both p53 and MDM2 are involved in the regulation of the apoptotic processes important for killing tumor cells by radiotherapy and chemotherapy.43 Thus, in patients carrying GG genotype and tumors with p53 mutations, the p53 functions were complementary inactivated. The inactivated p53 makes tumor cells more aggressive29 and poor response to treatments. MDM2 SNP 309 G allele has a modifying effect on the association between p53 and DFS in OSCC patients. The combined effects of the MDM2 genotypes and tumor p53 status on OSCC will require further confirmation in the investigation of other cancers and populations. Further understanding of the interactions between p53 and MDM2 and their regulatory mechanisms is critical for discovering new therapies for managing human cancers. Specifically, targeted inhibition of the p53/MDM2 interaction might be a promising avenue for future cancer treatment.44, 45 Conflict of Interest Statement None declared. 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45. 45Chene P. Inhibition of the p53-MDM2 interaction: targeting a protein–protein interface. Mol Cancer Res. 2004;2:20–28. MEDLINE a Department of Otolaryngology, Head and Neck Surgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan b Graduate Institute of Clinical Medical Science, Chang Gung University, Tao-Yuan, Taiwan c Division of Hematology/Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan d Department of Public Health, National Defense Medical Center, Taipei, Taiwan e Department of Public Health, Chang Gung University, 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan 333, Tao-Yuan, Taiwan f Taipei CGMH Head and Neck Oncology Group, Tao-Yuan, Taiwan  Corresponding author. Address: Department of Public Health, Chang Gung University, 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan 333, Tao-Yuan, Taiwan. Tel.: +886 3 2118800x5125; fax: +886 3 2118700.
PII: S1368-8375(08)00091-2 doi:10.1016/j.oraloncology.2008.03.006 © 2008 Elsevier Ltd. All rights reserved. | |
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