DUSP2 methylation is a candidate biomarker of outcome in head and neck cancer

Introduction

Despite improving clinical outcomes, optimal protocols for administration of combined modality therapy and biomarkers to inform the optimal use of such therapy have not been definitively identified in head and neck squamous cell carcinoma (HNSCC) (1). We have previously shown that mutations and polymorphisms in TP53 and in MDM2 predict response and survival in HNSCC patients treated with platinum-based chemoradiotherapy (CRT) (2-4).

The dual specificity phosphatases (DUSPs) comprise a large family of genes encoding enzymes which catalyse the dephosphorylation of serine, threonine and tyrosine residues in different types of mitogen-activated protein kinases within the MAPK TXY (Thr-Xaa-Tyr) motif (5). DUSP2 is an inducible nuclear phosphatase highly expressed in activated immune cells (6,7).

DUSP2 acts as a tumour suppressor at least in part via its physiological substrate ERK2 (8,9). Phosphorylation of ERK1/2, via Ras/Mek/ERK pathway, activates cell proliferation and survival (10-13) in response to a wide range of stimuli, including radiation, hypoxia and chemotherapeutic agents (14).

Silencing of DUSP2 leads to prolonged activation of the ERK pathway (15) that in turn has been associated with many aspects of tumour phenotype (16-18). Nonetheless DUSP2 has been involved in the initiation and development of acute leukaemia (19) and in the pathogenesis of human solid cancers, where a loss of its expression has been associated with breast, colon, lung, ovary, kidney and prostate tumours in hypoxic conditions (8,15). The strong relationship between hypoxia and downregulation of DUSP2 affords a mechanistic link with angiogenesis and metastasis (20).

Overexpression of EGFR, leading to proliferation, angiogenesis and metastasis is frequently observed in HNSCC (21), but correlation with hypoxia is still debated (22,23). DUSP2 affects the sensitivity of cancer cells to chemotherapy in vitro (15). Moreover, DUSP2 is a transcriptional target of wild-type p53 (24) a determinant of treatment response in HNSCC (25).

In the present study, we have tested the hypothesis that DUSP2 is transcriptionally silenced by methylation in HNSCC and in turn investigated the role of DUSP2 as determinant of clinical outcome in locally advanced head and neck squamous cell carcinoma (LA-HNSCC) treated with platinum-based CRT.

Methods

Cell lines

Five human HN cancer cell lines (CAL27, CAL33, HEp-2, HNO41, HNO91) were used to evaluate DUSP2 expression and aberrant methylation in the CpG island. Cells were routinely cultured as previously described (26).

Gene expression analysis

Real-time qRT-PCR was performed on RNA extracted from cell lines using the RNeasy Mini Kit (Qiagen, Crawley, West Sussex, UK) according to standard procedures. DUSP2 expression was assessed by TaqMan PCR assay Hs00358879_m1 using the ABI-Prism 7000 Sequence Detection System (Life Technologies, Carlsbad, California, US). Beta-2-microglobulin (Hs99999907_m1) was used as a control housekeeping gene.

Clinical cases and molecular analyses

Formalin fixed paraffin embedded (FFPE) specimens were obtained at diagnosis from 129 LA- HNSCC patients (stage III and IV) in Cuneo, from 1998 to 2013.

The distribution of gender, age, performance status (PS), tumor size (T), lymph nodes (N), histological grade (G), smoking habit and primary sites is shown in Table 1.

Table 1 Characteristics of LA-HNSCC patients (N=129) and tumours
Full table

Smokers were defined according to Ang et al. [2010] (27) with a cut-off of 10 pack-year.

Mutational analysis of TP53 and genotyping of the SNP Pro72Arg (rs1042522) were performed as previously described (2-4). TP53 mutations were classified according to the IARC TP53 mutation database (http://wwwp53.iarc.fr/), the Poeta et al. [2007] (28) study and the Evolutionary Action score of TP53 (EAp53) (http://mammoth.bcm.tmc.edu/EAp53/) (29). EGFR was quantified in 72 patients by IHC as already described (30).

HPV was searched by DNA-PCR using specific primer pairs for type 16 in 129 patients’ tissues; IHC staining for p16 (31) was performed in 118 patients’ tissues. This work was carried out in accordance with the Declaration of Helsinki. The study and the informed consent for biological samples collection and research proposal, obtained from patients, were approved by the Ethical Committee of S. Croce & Carle Teaching Hospital in Cuneo (approval n. 198/13).

Methylation analysis

DNA samples were extracted from cell lines and FFPE tissues using standard protocols. In particular, genomic DNA was purified by proteinase K digestion of 10 m sticks cut from paraffin sections using xylene–phenol protocol, as previously described (3).

DUSP2 CpG island methylation was analyzed by pyrosequencing (Biotage, Uppsala, Sweden). Following PCR primers were designed to amplify a fragment of 134 bp, covering part of IVS I and exon 2: F 5'-GTAGATAGGAGTTTTGGAGT-3'; R5'-BIOT-CTCTTCCCCTCCTTACAAA-3'. 500 ng genomic DNA were amplified and analysed by QCpG Software (Qiagen) as already reported (32).

We used a universal commercial human DNA (CpGenome Universal Methylated DNA, Millipore Corporation, Billerica, MA, USA) as methylated (met) control (average methylation 98%), while DNA obtained from a pool of 5 healthy head and neck epithelia was employed as unmethylated (unmet) control [mean value of DUSP2 CpG island methylation was 4% (range, 3–7%)]. On the basis of this consideration we established a methylation cut-off of 10%.

Statistical analysis

Relationships between DUSP2 gene methylation, clinical (gender, tumour site, PS, T, N, G, smoke and clinical response) and molecular (SNP rs1042522 and TP53 sequence) characteristics were analysed by cross-tabulation.

OS analysis was based on the time from diagnosis to death or last contact in which the survivors were censored. PFS analysis was based on the time from diagnosis to first event (loco-regional recurrence or distant metastasis); patients without an event were censored at their last follow-up.

OS and PFS curves were calculated using the Kaplan-Meier method, where statistical significance of each variable was tested with log-rank test.

Univariate analysis was performed on each variable, then for DUSP2/EGFR (N=72) and DUSP2/TP53 (N=101), and lastly in 70 DUSP2/EGFR/TP53 patients’ combination.

Significant variables identified in the univariate analysis with P≤0.05 entered into a multivariate analysis performed on 70 DUSP2/EGFR/TP53 patients. Same analysis was repeated considering variables with P<0.20. The level of significance was P<0.05. Data are presented as hazard ratios (HRs) with 95% confidence intervals (CIs). Statistical analyses were performed using SPSS version 13 (SPSS, Chicago, IL, USA) program.

Results

Methylation-dependent transcriptional silencing of DUSP2 in HNSCC cell lines

Analysis of DUSP2 expression by qRT-PCR showed that the mRNA was undetectable in HNO91 and CAL33, low in HNO41 and CAL27 and high in HEp-2 cell lines. Pyrosequencing revealed that the DUSP2 CpG island was unmethylated in HEp-2 cells and methylated in the other cell lines, with a clear correlation between methylation and DUSP2 down-regulation (Figure S1).

Figure S1 Methylation-dependent transcriptional silencing of DUSP2 in HNSCC cell lines. (A) Expression and methylation analysis of DUSP2 in HNSCC cell lines; (B) DUSP2 methylation profile of HNSCC cell lines. The level of methylation by pyrosequencing is proportional to the degree of shading in the circles, which represent individual CG dinucleotide in the amplified fragment; (C) representative programs showing HEp2 (DUSP2-unmet) and CAL33 (DUSP2-met) cells. DUSP2, dual-specificity-phosphatase-2; HNSCC, head and neck squamous cell carcinoma; CG, Cytosine Guanine.

Methylation analysis in primary LA-HNSCC tissues

We performed pyrosequencing analysis of DUSP2 CpG island methylation in a well-annotated series of 112 cases LA-HNSCC from our clinical practice. We excluded patients with obscure cancers (N=3) and HPV16 positive oropharynx tumours (N=14). Clinico-pathological details of the cases are shown in Tables 2 and 3. Using 10% mean methylation through methylation variable sites of the amplified fragment of the CpG island as a cut-off, 51/112 (45.5%) of cases were deemed positive for DUSP2 methylation. Methylation positivity was more frequent among patients with small tumour size (T1/T2) (25/51=49.0% methylated vs. 9/61=14.8% unmethylated; P<0.0001), but there was no significant correlation between methylation status and gender, primary tumour site, PS, N, G, smoke and clinical response. Furthermore, there was no association between DUSP2 methylation and TP53 mutational status or the rs1042522 polymorphism.

Table 2 Distribution of methylated and unmethylated DUSP2 in tissues at diagnosis (N=112) according to clinicopathological characteristics of patients
Full table

Table 3 Distribution of methylated and unmethylated DUSP2 in tissues at diagnosis (N=112) according to clinicopathological characteristics of patients
Full table

DUSP2 methylation in combination with TP53 status predicts outcome in LA-HNSCC

We asked if methylation of DUSP2 is a biomarker of clinical outcome in LA-HNSCC treated with CRT, by analysis of time-dependent end points in cases positive or negative for DUSP2 methylation. There was no difference between cases positive (N=49) and negative for DUSP2 methylation (N=55) in either OS (P=0.64) nor PFS (P=0.76).

Since DUSP2 is a transcriptional target for p53, we next asked whether TP53 and DUSP2 status together might influence outcomes in the 101 patients for which both parameters were available. We identified 59 mutated patients (58.4%), with 46 missense (78%) and 13 deletions with frameshift effects (22%). In univariate analysis, no difference in OS or PFS was found among patients carrying low (N=22) vs. high (N=24) missense mutations vs. deletions (N=13), according to EAp53 analysis (29). The same result was obtained when comparing patients carrying disruptive (N=27) vs. non-disruptive (N=32) mutations, according to Poeta et al. [2007] (28). This allowed us to consider the 59 patients with TP53 mutation as a unique group (TP53-mut) in subsequent analyses. OS was longer in TP53-mut vs. TP53-wt (N=42) cases (P=0.03) (Figure 1A). This was not observed in PFS.

Figure 1 Kaplan-Meier curves for TP53 status (N=101) and combination for DUSP2-unmet/TP53 status (N=54). (A) LA-HNSCC patients (N=101) carrying TP53-mut (N=59) showed a longer OS (months) compared to patients with TP53-wt (N=42) (P=0.03, median 60 vs. 25.5 months for mut and wt patients, respectively); (B) in LA-HNSCCs with DUSP2-unmet tissues (N=54) TP53-mut patients (N=29) showed a longer OS (P=0.012), given in months, compared to TP53-wt ones (N=25) with a median of 80 and 24 months for mut and wt respectively. LA-HNSCC, locally advanced head and neck squamous cell carcinoma; DUSP2, dual-specificity-phosphatase-2.

Combining TP53 with DUSP2 (N=101) and stratifying for TP53 status, we found that among the 54 unmet-DUSP2 cases, those carrying TP53 mutations (29/54; 53.7%) showed a longer OS (P=0.012), compared to cases with TP53-wt (25/54; 46.3%) (P=0.064) (Figure 1B). Again, no difference was found in PFS. Instead, in the 47 met-DUSP2 patients, the stratification for TP53 didn’t show any significant correlation.

DUSP2 methylation status predicts outcome in LA-HNSCC when combined with EGFR

Cases with low EGFR (N=19) had better outcome compared to patients with high-EGFR expression (N=53) although this was not significant (P=0.42). Combining EGFR with DUSP2 methylation status, we observed that cases with low EGFR and unmethylated DUSP2 (N=10) had longer OS than those with low-EGFR and met-DUSP2 (N=9) (P=0.013) (Figure 2A). On the contrary, in cases with high EGFR expression (N=53), no difference was seen in OS, although there was a trend suggesting this (Figure 2B). There was no difference in PFS in any EGFR/DUSP2 combination.

Figure 2 Kaplan-Meier curves for EGFR expression combination with DUSP2-unmet (N=19) and DUSP2-met (N=53) patients. (A) Among LA-HNSCCs with low-EGFR expression (N=19) DUSP2-unmet patients (N=10) showed a better OS (P=0.013), given in months, compared to DUSP2-met ones (N=9) with a median of 123 vs. 56 for unmet and met tissues, respectively; (B) an opposite trend was seen for LA-HNSCCs with high-EGFR expression (N=53) in which patients with met-DUSP2 tissues (N=17) showed a numerical longer OS (months) compared with unmet-DUSP2 ones (N=36) (P=0.15; median 83 vs. 24 months for met and unmet, respectively) although this difference was not significant. LA-HNSCC, locally advanced head and neck squamous cell carcinoma; DUSP2, dual-specificity-phosphatase-2.

Multivariate analysis for DUSP2/EGFR/TP53 combination markers

Finally we tested the utility of the DUSP2/EGFR/TP53 triple combination in predicting clinical outcome in the 70 patients characterised for all those markers. The cumulative P value resulted significant (P=0.005). DUSP2-unmet/high-EGFR/TP53-wt makeup (N=20) was identified as the major risk factor associated with shorter OS (P=0.0007) (Figures 3,4).

Figure 3 DUSP2-unmet/high-EGFR/TP53-wt makeup (N=20) is the major risk factor associated with shorter OS (P=0.0007), given in months, identified in our cohort of CRT-treated LA-HNSCC patients. LA-HNSCC, locally advanced head and neck squamous cell carcinoma; DUSP2, dual-specificity-phosphatase-2.

Figure 4 Univariate analysis (A) and logistic regression model (B) and (C) in the 70 DUSP2/EGFR/TP53 patients.

Instead, patients with DUSP2-met/high-EGFR/TP53-wt (N=4) showed the longest OS and the highest HR, although not significant, due to their small number (Figure 4A,B,C).

Discussion

In the present study we have identified DUSP2, a negative regulator of MAP kinases (6), as a novel gene subject to methylation-dependent transcriptional silencing in LA-HNSCC and we show that the quantitative level of DUSP2 methylation, when combined with EGFR expression and TP53 mutational status, has utility as a candidate biomarker of clinical outcome in patients treated with CRT. To the best of our knowledge, this is the first demonstration that CpG island methylation regulates DUSP2 expression in HN cell lines and it is methylated in clinical cases, with 45% of cases in our LA-HNSSC series positive for methylation at diagnosis. Some studies have previously reported the epigenetic inactivation of DUSP2 in human cancer cell lines (wherein methylation is associated with transcriptional silencing) but not in clinical cases (33).

In vitro, expression of DUSP2 induces apoptosis, inhibits tumour growth and abolishes hypoxia-induced drug resistance (15). These biological properties are all features of tumour suppressor genes and our demonstration of methylation-dependent transcriptional silencing of DUSP2 in LA-HNSCC affords further experimental evidence in support of this hypothesis. DUSP2 expression is a determinant of cellular sensitivity to some cytotoxic agents, including cisplatin (15) and targeted therapies such as lapatinib (34). Although DUSP2 mRNA down-regulation occurs in a number of solid tumours, increased expression levels of DUSP2 predict a worse OS in serous ovarian carcinoma (35). This apparent contradiction has recently been reported in other potential tumour-suppressor genes (36) including DUSP1 (7) and DUSP6 (37).

Low expression levels of DUSP2 correlates with reduced relapse-free survival in ERBB2-positive breast cancer patients (34), prompting us to examine DUSP2 methylation in combination with EGFR. Surprisingly, we found that LA-HNSCC cases with DUSP2 methylation (and therefore likely expressing low levels of DUSP2) and high-EGFR had increased OS compared with patients with DUSP2 unmethylated cases (although this trend did not reach significance), This effect might be associated with a favourable effect of MAPKs that are not inhibited by DUSP2 {Givant-Horwitz et al. [2004]} (35). Instead, low-EGFR patients with DUSP2-met tissues showed a shorter OS compared to patients with DUSP2-unmet tissues. Our interest in DUSP2 originated from studies showing that it is also a direct transcriptional target of wild-type p53 and may participate in the TP53-dependent DNA damage response to oxidative stress (24). Although some data suggest that TP53 might be used to predict outcome in HNSCC, its analysis has not become part of the routine evaluation in clinical settings and its role as a prognostic marker remains controversial (38).

In our cohort of LA-HNSCC, TP53 was the only biomarker, among those analysed, able to independently predict longer OS. Interestingly, we observed longer OS in cases with wild-type TP53 (58%) compared with mutant TP53. This is in apparent contradiction with outcome of CRT as suggested by others (27). Nonetheless, stratifying cases by DUSP2 methylation status, we observed that those with DUSP2 methylation and wild-type TP53 showed a numerical improved OS compared to TP53 mutant, while, in DUSP2 unmethylated cases, those with mutant TP53 showed longer OS.

Mechanistically, it might be speculated that the reduced expression of DUSP2 induces both STAT3 and MAPKs activation in tumours with intact TP53, while the consequence of normal expression of DUSP2 in mutated TP53 cases may increase cellular sensitivity to CRT (39,40).

When we analysed together the markers DUSP2/EGFR/TP53, we found that DUSP2 silencing at diagnosis was associated with more favourable clinical outcomes when combined with EGFR overexpression and wild-type TP53-wt. This highlights the relevance of intact p53 activity for the suppression of HNSCC development.

In conclusion, we show that DUSP2 methylation might have utility in predicting OS of CRT-treated LA-HNSCC patients suggesting that it may have utility as a biomarker of response to CRT therapies.

DUSP2 function in cancer supports the need for larger clinical studies to further investigate the role of this molecule.

Acknowledgements

We would like to thank Gerard Milano from Centre Antoine Lacassagne (Nice, France) for CAL27 and CAL33. This work has been partially supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Foundation (to CLN and MCM) and ARCO (Attività Ricerca Clinica Oncologica) Foundation.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: The study and the informed consent for biological samples collection and research proposal, obtained from patients, were approved by the Ethical Committee of S. Croce & Carle Teaching Hospital in Cuneo (approval No. 198/13).

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