Dual specificity phosphatase 6 as a predictor of invasiveness in papillary thyroid cancer

in European Journal of Endocrinology
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  • 1 Department of Pathology, Department of Pathology, Department of Internal Medicine, Department of Internal Medicine, St Mary's Hospital, the Catholic University of Korea, 64 Daeheung-ro, Chungku Daejeon 301-723, Korea

(Correspondence should be addressed to M Shong; Email: minhos@cnu.ac.kr); Y S Jo; Email: ysmrj@cnuh.co.kr)

Objective

The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC.

Methods

To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAFV600E mutation.

Results

The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAFV600E mutation. However, the expressions of c-Fos and PCNA were elevated in BRAFV600E-positive PTC compared with those in BRAFV600E-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells.

Conclusions

The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAFV600E mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

Abstract

Objective

The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC.

Methods

To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAFV600E mutation.

Results

The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAFV600E mutation. However, the expressions of c-Fos and PCNA were elevated in BRAFV600E-positive PTC compared with those in BRAFV600E-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells.

Conclusions

The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAFV600E mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

Introduction

Since activating mutations in BRAF kinase were discovered in cancers, tremendous efforts have been made to determine the precise mechanism of carcinogenesis induced by BRAFV600E, which is frequently observed in melanoma and papillary thyroid cancer (PTC) (1). The BRAFV600E mutation induces constitutive phosphorylation of MEK1/2 (MAPK kinase) (2). The activated MEK1/2, dual specific ERK1/2 (extracellular signal-regulated kinase, MAPK, ERK) kinases phosphorylate the threonine 202 and tyrosine 204 residues of ERK1/2 (3). The activated ERK1/2 translocates to the nucleus and activates the transcription of a specific set of genes required for malignant transformation in NIH3T3 cells and in thyroid-specific BrafV600E transgenic mice (1, 4). Inappropriate activation of the MEK-ERK signaling pathway has been regarded as a common carcinogenic pathway activated by RET/PTC rearrangements and the BRAFV600E mutation. On the basis of these findings, novel therapeutic agents are under investigation to block the MEK-ERK signal pathways for the treatment of advanced thyroid cancer (5, 6, 7, 8).

The phosphorylation and dephosphorylation cascade of ERK1/2 is a critical process for the response of cells to mitogens or to prevent the deleterious effects of prolonged stimulation. This failsafe mechanism is mainly mediated by the activity of phosphatases such as MAPK phosphatases (MKPs) (5, 6, 7). In particular, dual specificity phosphatase 6 (DUSP6), also known as MKP3, specifically inactivates ERK1/2 and is a potential negative regulator of fibroblast growth factor (FGF)-induced ERK activation (8). Moreover, gene expression analyses showed that DUSP6/MKP3 was markedly increased in melanoma cell lines harboring BRAFV600E or NRASQ61R mutations (9). However, the precise role of phosphatases remains to be elucidated and the expression pattern of DUSP6 has not been investigated in tissues derived from BRAFV600E-positive PTC (10).

In this study, ERK activity in PTC was assessed by immunohistochemical (IHC) staining using anti-phospho-p44/42MAPK, anti-p44/42MAPK, anti-DUSP6, anti-c-Fos, anti-c-Myc, and anti-cyclin D1 antibodies. Furthermore, the expression of DUSP6 and the relationship between ERK1/2 and DUSP6 were investigated. The current results showed no clear relationship between ERK phosphorylation and the BRAFV600E mutation. Interestingly, DUSP6 expression was markedly increased in PTC compared with that in benign neoplasms, and increased DUSP6 expression was correlated with high ERK staining intensity and high-risk biological features.

Materials and methods

Selection of patients and analysis of clinicopathological data

Thyroid tissue specimens were obtained from 209 patients (42 benign neoplasms and 167 PTCs) who underwent surgery from 2004 to 2005 at the Center for Endocrine Surgery, Chungnam National University Hospital, Daejeon, Korea. Benign neoplasms were composed of 28 nodular hyperplasias and 14 follicular adenomas. Patient information and clinicopathological parameters were analyzed retrospectively. The age and sex distribution are summarized in Table 1. The tumor node metastasis classification of the International Union Against Cancer (UICC) was used for staging PTC samples. All protocols were approved by the institutional review board.

Table 1

Baseline characteristics and expression patterns of pERK, ERK, DUSP6, c-Fos, c-Myc, cyclin D1, and PCNA in benign neoplasms and papillary thyroid carcinoma.

Benign neoplasmsPapillary cancerTotalP value
Cases (n)42167209
Age (years)47.1±11.143.5±12.60.09a
Sex
 Male10 (23.8%)41 (24.6%)510.9b
 Female32 (76.2%)126 (75.4%)158
Size (cm)2.4±0.82.7±10.07a
pERK (20G11)c
 Grade 035 (85.4%)159 (95.2%)1940.005d
 Grade 14 (9.8%)8 (4.8%)12
 Grade 22 (4.9%)02
 Grade 3000
pERK (D13,14,4e)c
 Grade 036 (87.8%)158 (94.6%)1940.026d
 Grade 13 (7.3%)9 (5.4%)12
 Grade 22 (4.9%)02
 Grade 3000
ERK
 Grade 0000<0.001d
 Grade 124 (58.5%)18 (10.3%)42
 Grade 213 (31.7%)113 (67.7%)144
 Grade 34 (9.8%)36 (21.6%)40
ERK localizatione
 Nucleus24 (58.5%)90 (55.2%)1140.7b
 Cytoplasm17 (41.5%)73 (44.8%)90
DUSP6f
 Grade 038 (90.5%)0 (0)38<0.001d
 Grade 14 (9.5%)20 (12.1%)24
 Grade 2072 (43.6%)72
 Grade 3073 (44.2%)73
c-Fos
 Grade 038 (90.5%)44 (26.3%)82<0.001d
 Grade 14 (9.5%)28 (16.8%)32
 Grade 2068 (40.7%)68
 Grade 3027 (16.2%)27
c-Myc
 Grade 034 (81.0%)57 (34.1%)91<0.001d
 Grade 18 (19.0%)30 (18.0%)38
 Grade 2065 (38.9%)65
 Grade 3015 (9.0%)15
Cyclin D1
 Grade 036 (87.8%)34 (20.4%)70<0.001d
 Grade 15 (12.2%)61 (36.5%)66
 Grade 2055 (32.9%)55
 Grade 3017 (10.2%)17
PCNA
 Grade 028 (66.7%)23 (13.8%)51<0.001d
 Grade 17 (16.7%)70 (41.9%)77
 Grade 27 (16.7%)61 (36.5%)68
 Grade 3013 (7.8%)13

Expression of pERK and ERK could not be determined in one case out of 42 benign neoplasms.

Data are presented as mean±s.d. and P values were calculated by independent sample t-tests.

P values were calculated by pair-wise comparisons from Pearson's χ2 test.

P values were calculated by comparisons of three or four groups from linear-by-linear associations.

ERK localization could not be determined in four cases of 167 PTCs.

Staining intensity of DUSP6 could not be determined in two cases out of 167 PTCs.

DNA isolation and dideoxy sequencing

Genomic DNA from paraffin-embedded thyroid tissue specimens was prepared from five 10 μm-thick sections after microdissection. In the case of cancers, the paraffin-embedded thyroid tissue specimens consisted of more than 90% tumor cells. The sections were mounted on slides and dried for 12 h at 37 °C, deparaffinized with xylene, and rehydrated in a series of graded alcohol solutions, followed by deionized water. Genomic DNA was isolated using the EZ1 DNA Tissue kit (Qiagen). Exon 15 of the BRAF gene in a 5 μl volume of genomic DNA was amplified by PCR using standard conditions (95 °C×5 min; 94 °C×30 s, 58 °C×30 s, and 72 °C×30 s for 32 cycles; 70 °C×10 min) and the following primers: forward 5′-ATGCTTGCTC TGATAGGAAA-3′ and reverse 5′-ATTTTTGTGA ATACTGGGGAA-3′. The amplified products were purified using the MinElute PCR Purification kit (Qiagen), and the purified PCR products were sequenced on an ABI PRISM 3730XL-automated capillary DNA sequencer by using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA).

Tissue microarray construction

Paraffin-embedded thyroid tissue samples were analyzed using a tissue array instrument (11). Briefly, representative areas of each tumor (both benign neoplasms and PTC) were selected and marked on the hematoxylin–eosin-stained slide, and its corresponding tissue block was sampled for the tissue microarrays. The designated zone of each donor block was punched with a 1 mm-diameter tissue cylinder, and the sample was transferred to a recipient block. Each sample was arrayed on duplicate blocks to minimize tissue loss.

IHC staining

IHC staining for phospho-p44/42 MAPK, p44/42 MAPK, DUSP6, DUSP4, c-Fos, c-Myc, cyclin D1, and PCNA was performed in 42 benign neoplasms and 167 PTCs. Briefly, 4 μm-thick tissue sections were heated at 60 °C, deparaffinized in xylene, and hydrated in a graded series of alcohol. When necessary, antigen retrieval was performed by microwaving in citrate buffer for 10 min. Endogenous peroxidase activity was inactivated by incubation in 3% hydrogen peroxide for 10 min. Nonspecific binding sites were blocked by incubating in 10% normal goat serum diluted with PBS. Tissue sections were then incubated for 60 min at room temperature, with the following primary antibodies: p44/42 MAPK rabbit mAb, phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) rabbit mAb, phospho-p44/42 MAPK (Thr202/Tyr204) (20G11) rabbit mAb (Cell Signaling Technology, Beverly, MA, USA), DUSP6 mouse mAb, DUSP4 mouse mAb (Abnova, Taipei, Taiwan), c-Fos rabbit pAb (K-25) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), c-Myc mouse mAb (9E10) (Santa Cruz Biotechnology, Inc.), cyclin D1 rabbit mAb (Dako, Carpinteria, CA, USA), PCNA mouse mAb (Invitrogen Corporation), phospho-MEK1/2 (Ser221) (166F8) rabbit mAb, and MEK1/2 (L38C12) mouse mAb (Cell Signaling Technology). All sections were sequentially treated with biotinylated antirabbit or antimouse immunoglobulin for 30 min, peroxidase-labeled streptavidin for 30 min, and diaminobenzidine in the presence of hydrogen peroxide. Controls were incubated with PBS instead of a primary antibody and no positive staining was observed. In addition to negative controls, positive controls were provided by sections of colon and breast carcinoma stained for phospho-p44/42 MAPK, breast carcinoma for p44/42 MAPK, human pancreas for DUSP6 and DUSP4, and human lung squamous cell carcinoma for c-Fos, c-Myc, cyclin D1, and PCNA. Staining was scored as follows: 0, no staining; 1, weak or focal staining; 2, moderate staining in most tumor cells; and 3, strong staining in most tumor cells. The tumor cells were considered immunopositive if the nucleus showed homogeneous staining for c-Fos, c-Myc, cyclin D1, and PCNA. Subcellular localization of p44/42 MAPK was categorized into two groups: nuclear localization of p44/42 MAPK when tumor cells showed nuclear staining alone or predominantly nuclear staining and cytoplasmic localization when tumor cells showed cytoplasmic staining alone or predominantly cytoplasmic staining.

Immunoblot analysis

The cells were lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 25 mM NaF, 10 mM β-glycerol phosphate (pH 7.5), 120 mM NaCl, 1% NP-40, and a protease inhibitor cocktail (Roche). The cell lysates were denatured via boiling for 5 min and separated using SDS–PAGE. After transfer to a nitrocellulose membrane (Amersham Biosciences), the membranes were blocked with 5% skim milk and incubated with primary and secondary antibodies overnight at 4 °C and for 1 h at room temperature respectively. The immune-reactive bands were developed using peroxidase-conjugated secondary antibodies (phototope-HRP Western Blot Detection Kit; New England Biolabs, Beverly, MA, USA).

Cell viability and scratch assay

SiDUSP6 and siCTL (Invitrogen) were purchased and used to treat FRO cells (BRAFV600E-positive thyroid cancer cell lines) with the manufacturer's protocols (12). After the 24-h treatment with SiDUSP6 and siCTL, the cells were plated in 96-well plates and an MTT solution (Sigma–Aldrich) was added to the plated cells at the indicated times. We measured the absorbance at 595 nm using an EMax precision microplate reader (Molecular Devices, Sunnyvale, CA, USA). Again, the SiDUSP6- and siCTL-treated FRO cells were plated in six-well plates at 5×105 cells/well. After 16 h, the cell surface in three places was scratched using a p200 pipette tip. The cells were observed after 12 h using an Olympus IX71 microscope (Olympus). The migration rates were calculated using the following equation: (full length−scratched length)/full length×100. The image analysis was performed using the Image J v1.42q software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Group comparisons of categorical variables were performed using the χ2 test or linear-by-linear association. The means were compared with the independent samples t-test, one-way ANOVA, or Mann–Whitney U test. All reported P values are two sided. Analyses were performed using SPSS Version 18.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

PTC tumor cells show barely detectable ERK phosphorylation but a significant increase in total ERK expression compared with benign neoplasms

Two rabbit mAbs derived from different clones were used for the detection of ERK1/2 phosphorylation by IHC analysis based on the recommendations of the manufacturer (D13.14.4E and 20G11; Cell Signaling Technology), and both antibodies showed similar staining patterns in our experiments. Positive staining of phospho-ERK1/2 could be easily observed in normal follicular cells, and stromal cells served as internal positive controls (Fig. 1A and B respectively). However, most PTC tumor cells showed weak or no staining for phospho-ERK1/2 (Fig. 1A), which was more frequently detected in benign neoplasms than in PTCs (Table 1). IHC patterns of total ERK1/2 in benign neoplasms and PTC were different from those of phospho-ERK1/2. Regarding total ERK1/2 staining, most benign neoplasms showed weak or focal staining intensity (90.2%), whereas 149 cases (89.3%) of PTC showed moderate or strong staining intensity (P<0.001). In the case of MEK1/2 phosphorylation, most of the tumor samples showed moderate staining scores (Supplementary Figure S1A, see section on supplementary data given at the end of this article). Stimulation of RAF-MEK-ERK signaling by growth factors results in ERK phosphorylation and nuclear translocation to increase ERK-dependent transcriptional output. We therefore analyzed the subcellular localization of ERK to assess its activation status and determine the functional impact of undetectable ERK phosphorylation. As shown in Table 1, the proportion of PTCs (55.2%) showing nuclear localization of ERK was not significantly different from that of benign neoplasms (58.5%). In summary, ERK phosphorylation was barely detectable in PTC and subcellular localization of ERK did not differ in benign neoplasms and PTCs, although a significant proportion of PTCs showed higher staining intensities of total ERK than did benign neoplasms (Table 1).

Figure 1
Figure 1

Representative figures of immunohistochemical staining for phospho-ERK1/2, ERK1/2, and DUSP6. (A) In normal follicular cells, phospho-ERK1/2 is mainly detected in the nucleus. (B) Phospho-ERK1/2 is undetectable in tumor cells, but surrounding normal follicular cells and stromal cells show high staining intensity of phospho-ERK1/2. (C and D) Tumor cells show markedly increased DUSP6 expression. DUSP6 is exclusively located in the cytoplasm. (E and F) Phospho-ERK1/2-positive cases show focal staining intensity in limited regions. (G) In these regions (E and F), DUSP6 expression is notably decreased. (H and I) Another case showing focal phospho-ERK staining intensity (H) arrows indicate decreased DUSP6 expression (I) in the phospho-ERK1/2 detectable area.

Citation: European Journal of Endocrinology 167, 1; 10.1530/EJE-12-0010

Tumor-specific expression of DUSP6 could be associated with undetectable ERK phosphorylation despite a marked increase in the expression of ERK-related genes in PTC

Most benign neoplasms (90.5%) did not show positive DUSP6 staining, whereas 145 (87.8%) cases of PTC showed moderate or strong cytoplasmic staining for DUSP6 (Fig. 1C and D). These differences in the pattern of expression of DUSP6 between the two groups reached statistical significance (P<0.001, Table 1). IHC analysis using an anti-DUSP4 antibody to verify the specificity of the anti-DUSP6 antibody revealed no immunoreactivity for DUSP4 in PTC (Supplementary Figure S2A and S2B, see section on supplementary data given at the end of this article). ERK1/2 phosphorylation was detectable in eight PTC cases, but even in these cases, a limited region of the tumor cells showed positive staining (Fig. 1E, F, and H). Interestingly, DUSP6 expression was remarkably decreased in these areas (Fig. 1G and I). In addition, when we performed the western blot analysis using the primary cultured BRAFV600E-positive PTC from two different patients (13), we could not easily detect ERK1/2 phosphorylation but detected DUSP6 (Supplementary Figure S3, see section on supplementary data given at the end of this article). These data strongly suggested that tumor-specific expression of DUSP6 might contribute to the undetectable levels of ERK phosphorylation.

To evaluate ERK-dependent transcriptional activity, MEK/ERK-related genes such as c-Fos (FOS), c-Myc (MYC), and cyclin D1 were detected by IHC analysis. Although phosphorylated ERK could not be detected in PTC, c-Fos, c-Myc, and cyclin D1 showed a statistically significant increase in PTC compared with that in benign neoplasms (all P values were below 0.001 and are shown in Table 1, Supplementary Figure S4, see section on supplementary data given at the end of this article). However, PCNA, which is not related to ERK activity, was also increased in PTC (P<0.001, Table 1, Supplementary Figure S4). The results of the statistical analysis of IHC data suggest that phosphorylated ERK might not be a molecular marker for the activity of ERK and ERK-related genes in PTC.

Increased ERK and DUSP6 expressions are distinct phenomena in PTC and unrelated to the BRAFV600E mutation status

On the basis of the results showing increased expression of ERK1/2, DUSP6, and ERK-related genes in PTC compared with that in benign neoplasms, a comparison of clinicopathological parameters and expression patterns of ERK1/2, DUSP6, and ERK-related genes according to absence or presence of BRAFV600E mutation was performed. Although the value of the BRAFV600E mutation as a prognostic factor has been suggested in large retrospective studies and meta-analyses, its specific prognostic impact is still controversial, particularly in eastern countries (14, 15, 16, 17). The present data did not show a correlation between the presence of the BRAFV600E mutation and high-risk biological features such as tumor size, extrathyroidal extension, multifocality, nodal metastasis, distant metastasis, and advanced stage at surgery (Table 2). Moreover, staining intensity and subcellular localization of ERK showed a similar pattern in the two groups (Table 2). Previous work suggested that various growth stimulations such as FGF or platelet-derived growth factor can induce DUSP6 expression through MEK-ERK-dependent pathways and, in turn, DUSP6 can regulate ERK activation (8, 18). In this study, the majority of PTC samples showed moderate-to-strong staining intensities for DUSP6 regardless of the BRAFV600E mutation status (Table 2). Interestingly, the immune reactivity of c-Fos and PCNA showed statistically significant differences according to the BRAFV600E mutation status (P<0.007 and P<0.016 respectively, Table 2). The expressions of the other two ERK-dependent genes, however, did not show differences between the two groups. Taken together, these data indicate that the tumor-specific expression of DUSP6 and ERK-related genes may be a response to persistent growth signals, which takes place in multiple carcinogenetic steps, rather than a BRAFV600E mutation-specific signaling pathway-dependent response.

Table 2

Clinicopathological characteristics and expression of pERK, ERK, DUSP6, c-Fos, c-Myc, cyclin D1, and PCNA according to the status of BRAFV600E mutation.

BRAFV600EmutationaBRAFV600E
Negative Positive P value
Number of cases43123
Age (years)41.7±14.644.1±11.90.29b
Sex
 Male14 (32.6%)26 (21.1%)0.13c
 Female29 (67.4%)97 (78.9%)
Tumor staging (T)
 T113 (30.2%)35 (28.5%)0.93d
 T223 (53.5%)68 (55.3%)
 T35 (11.6%)15 (12.2%)
 T42 (4.7%)5 (4.1%)
Tumor size (cm)2.5±0.82.6±1.00.09b
Extrathyroidal extension
 Negative21 (48.8%)67 (54.5%)0.52c
 Positive22 (51.6%)56 (45.5%)
Multifocality
 Negative35 (81.4%)96 (78%)0.64c
 Positive8 (18.6%)27 (22%)
Nodal metastasis
 Negative16 (37.2%)62 (50.4%)0.14c
 Positive27 (62.8%)61 (49.6%)
Distant metastasis
 Negative41 (97.6%)123 (100%)0.09c
 Positive1 (2.4%)0
TNM stage
 I33 (76.7%)88 (71.5%)0.834d
 II2 (4.7%)5 (4.1%)
 III6 (14.0%)25 (20.3%)
 IV2 (4.7%)5 (4.1%)
pERK staining intensity
 20G11
    Grade 038 (88.4%)120 (97.6%)0.028c
    Grade 15 (11.6%)3 (2.4%)
 D13,14,4e
    Grade 038 (88.4%)119 (96.7%)0.051c
    Grade 15 (11.6%)4 (3.3%)
ERK staining intensity
 Grade 14 (9.3%)14 (11.4%)0.91d
 Grade 230 (69.8%)82 (66.7%)
 Grade 39 (20.9%)27 (21.9%)
ERK localizatione
 Nucleus20 (48.8%)69 (57%)0.51c
 Cytoplasm21 (51.2%)52 (43%)
DUSP6 staining intensityf
 Grade 0000.29d
 Grade 17 (17.1%)13 (10.6%)
 Grade 218 (43.9%)54 (43.9%)
 Grade 316 (39%)56 (45.5%)
c-Fos staining intensity
 Grade 019 (44.2%)24 (19.5%)0.007d
 Grade 18 (18.6%)21 (17.1%)
 Grade 210 (23.3%)58 (47.2%)
 Grade 36 (14.0%)20 (16.3%)
c-Myc staining intensity
 Grade 021 (48.8%)35 (28.5%)0.096d
 Grade 16 (14.0%)24 (19.5%)
 Grade 212 (27.9%)53 (43.1%)
 Grade 34 (9.3%)11 (8.9%)
Cyclin D1 staining intensity
 Grade 012 (27.9%)22 (17.9%)0.576d
 Grade 114 (32.6%)47 (38.2%)
 Grade 213 (30.2%)41 (33.3%)
 Grade 34 (9.3%)13 (10.6%)
PCNA staining intensity
 Grade 012 (27.9%)11 (8.9%)0.016d
 Grade 114 (32.6%)56 (45.5%)
 Grade 213 (30.2%)47 (38.2%)
 Grade 34 (9.3%)9 (7.3%)

Presence or absence of BRAFV600E could not be determined in one case of PTC.

Data are presented as mean±s.d. and P values were calculated by independent sample t-tests.

P values were calculated by pair-wise comparisons from Pearson's χ2 test.

P values were calculated by comparisons of three or four groups from linear-by-linear associations.

ERK localization could not be determined in two cases of BRAFV600E-negative PTC and two cases of BRAFV600E-positive PTC.

Staining intensity of DUSP6 could not be determined in two cases of BRAFV600E-negative PTC.

DUSP6 might play a role in tumor progression and have a prognostic value in patients with PTC

On the basis of the increased DUSP6 expression in PTC and the lack of association with BRAFV600E mutation status, we investigated the impact of this phosphatase on high-risk biological features. Interestingly, certain prognostic factors were related to DUSP6 expression. Increased age was correlated with DUSP6 staining intensities (P=0.05, Table 3), and tumor size at operation also increased in correlation with DUSP6 staining intensity (P=0.01, Table 3). Finally, extrathyroidal extension was detected more frequently in association with increased DUSP6 staining intensity (P=0.02, Table 3). Because these clinicopathological data suggested the poor prognostic impact of DUSP6, we suspected that DUSP6 might play a role in tumor progression and have prognostic value in patients with PTC.

Table 3

Impact on prognostic markers and relationship between ERK expression and DUSP6.

DUSP6 staining intensity BRAFV600E positive
Grade 1 (n=20)Grade 2 (n=72)Grade 3 (n=73)P value
Age (years)39.5±10.242.3±13.546.1±11.70.05a
Sex (male:female)4:1616:56 20:53 0.4b
Tumor staging (T)
 T16 (30.2%)17 (23.6%)25 (34.2%)0.25b
 T210 (50%)43 (59.7%)37 (50.7%)
 T32 (10%)9 (12.5%)9 (12.3%)
 T42 (10%)3 (4.2%)2 (2.7%)
Tumor size (cm)2.1±0.82.7±0.92.8±1.00.01a
Extrathyroidal extension
 Negative13 (65%)43 (59.7%)31 (42.5%)0.02b
 Positive7 (35%)29 (40.3%)42 (57.5%)
Multifocality
 Negative18 (90%)54 (75%)58 (79.5%)0.62b
 Positive2 (10%)18 (25%)15 (20.5%)
Nodal metastasis
 Negative13 (65%)28 (38.9%)36 (49.3%)0.69b
 Positive7 (35%)44 (61.1%)37 (50.7%)
Distant metastasis
 Negative20 (100%)71 (100%)72 (98.6%)0.32b
 Positive001 (1.4%)
TNM stage
 I17 (81.0%)54 (75.0%)50 (68.5%)0.438b
 II1 (4.8%)2 (2.8%)4 (5.5%)
 III1 (4.8%)13 (18.1%)17 (23.3%)
 IV2 (9.5%)3 (4.2%)2 (2.7%)
pERK staining intensity
 20G11
    Grade 019 (95%)67 (93.1%)72 (98.6%)0.2b
    Grade 11 (5.0%)5 (6.9%)1 (1.4%)
 D13,14,4e
    Grade 019 (95%)67 (93.1%)71 (97.3%)0.4b
    Grade 11 (5.0%)5 (6.9%)2 (2.7%)
ERK staining intensity
 Grade 12 (10%)11 (15.3%)4 (5.5%)0.04b
 Grade 215 (75%)49 (68.1%)48 (65.8%)
 Grade 33 (15%)12 (16.7%)21 (28.8%)
ERK localizationc
 Nucleus10 (50%)42 (59.2%)38 (52.8%)0.86b
 Cytoplasm10 (50%)29 (40.8%)34 (47.2%)

Data are presented as mean±s.d. and P values were calculated by one-way ANOVA.

P values were calculated by comparisons of 3 or 4 groups from linear by linear associations.

ERK localization could not be determined in one case in the grade 2 group and one case in the grade 3 group.

Next, we performed cell viability and scratch assays with DUSP6 silencing to investigate whether DUSP could affect tumor cell behaviors in vitro (Fig. 2A). Although the phosphorylation of ERK was increased in the siDUSP6-treated FRO cells, we observed the significantly decreased cell viability of the siDUSP6-treated FRO cells compared with siCTL-treated FRO cells (Fig. 2B). Furthermore, in the scratch assay, siDUSP6 significantly affected the cell migration rate compared with siCTL (98.8±1.3 vs 47.8±8.3% respectively, P=0.009, Fig. 2C and D). These in vitro experiments supported that DUSP6 has a role in determining aggressive tumor behaviors.

Figure 2
Figure 2

DUSP6 silencing affected tumor cell migration. (A) The siDUSP6 treatment markedly decreased the DUSP6 expression compared with the siCTL treatment of the FRO cells. (B) The MTT assay demonstrating the cell viability of the siDUSP6- and siCTL-treated FRO cells. (C and D) The scratch assays were performed using the siDUSP6- and siCTL-treated FRO cells. Average means were compared with Mann–Whitney U test. All the reported P values are two sided. *P<0.05; **P<0.01. The data represent the mean±s.d. of three independent experiments.

Citation: European Journal of Endocrinology 167, 1; 10.1530/EJE-12-0010

Discussion

The MEK-ERK signaling cascades regulate critical steps required for normal cell proliferation and survival. Recently, the constitutive activation of the signaling components of this system has been regarded as a distinct carcinogenesis model in cancers associated with Ras, BRAF, and epidermal growth factor receptor mutations (19, 20, 21, 22). The active mutant BRAFV600E phosphorylates and activates MEK1/2, which in turn causes phosphorylation and nuclear translocation of ERK1/2 and subsequently leads to inappropriate cell cycle progression. Therefore, the MEK-ERK signaling pathway is a therapeutic target in the design of anticancer drugs for BRAFV600E-positive tumors. New drugs for the treatment of melanoma are currently in the clinical trial stage (23, 24).

Recent studies on melanoma showed that the intracellular levels of phospho-ERK1/2 did not correlate with the BRAFV600E mutation status (25, 26). Thus, intracellular levels of phospho-ERK1/2 might not reflect the activity of the BRAFV600E-MEK-ERK pathway. To exclude the nonspecific detection of phosphorylated ERKs, we selected two antibodies that were produced by different clones for the specific detection of phosphorylated ERKs in BRAFV600E-positive cells using western blot analysis (data not shown). The detection of phospho-ERK1/2 in normal follicular cells and stromal cells adjacent to the tumor suggested that the antibodies used in this study possessed adequate sensitivities and specificities. The specificities of the antibodies were further supported by consistent findings that the immunoreactivities for total ERK were mainly confined to the cytoplasm in tissues with no detectable phosphorylated ERKs.

To determine the activity of the MEK-ERK signaling pathway, we performed an additional IHC study using antibodies against ERK-dependent transcription factors in benign neoplasms and PTCs. The immunoreactivities for ERK, c-Fos, c-Myc, and cyclin D1 were more elevated in PTC than in benign neoplasms regardless of the BRAFV600E mutation status. In addition, c-Fos and PCNA were significantly elevated in BRAFV600E-positive PTC compared with BRAFV600E-negative PTC, whereas the expressions of c-Myc and cyclin D1 did not differ between the two groups. These data provided new insights on carcinogenesis in PTC. First, although phospho-ERK was barely detected, PTC might have persistent ERK-dependent transcriptional activity. Secondly, certain ERK-related genes such as c-Myc and cyclin D1 might be linked to various growth signals rather than the BRAFV600E-specific signaling pathway. Thirdly, a non-ERK-related gene, PCNA, was also upregulated in BRAFV600E (+) PTC, suggesting that ERK-independent signaling pathways may be involved in BRAFV600E-derived carcinogenesis.

The most significant finding of this study is the role of DUSP6 in PTC. Zuo et al. demonstrated that activated/phosphorylated ERK1/2 assessed by IHC analysis was not detected in most tumor tissues from melanoma and PTC patients with positive BRAFV600E mutation status. From these findings, they suggested that the absence of phospho-ERK1/2 in BRAFV600E tumors is mainly due to feedback inhibitory mechanisms (27). On the other hand, a recent study suggested that mutant BRAF kinase might be resistant to feedback inhibition by the corresponding phosphatase DUSP6/MKP3. In this study, increased activity of ERK-related genes was associated with an increase in the levels of DUSP6. These data suggest that the phosphatase activity of DUSP6 might be crucial for rapid and transient activation of the ERK signaling pathway to elicit cell proliferation and maintain high steady-state levels of phospho-ERK (28). Because the high level of total ERK is correlated with DUSP6/MKP3 expression levels, DUSP6/MKP3 may be responsible for ERK reactivation rather than playing a role in feedback inhibition. In fact, phosphorylation–dephosphorylation cycles of ERK might be a critical step in ERK activation as an essential process in the response of the cell to various growth signals (29, 30). A recent systems biology approach suggested that feedback regulation of DUSP6/MKP3 might control the kinetics and the extent of ERK activation (31). The magnitude and duration of ERK activation are critical for cellular responses and modulated by kinases and phosphatases. A mathematical model suggested that kinases control signaling amplitude rather than the duration of signaling, whereas phosphatases regulate the duration of signal propagation (32, 33, 34). On the basis of this concept, signal propagation driven by BRAFV600E might be controlled by ‘dephosphorylation of ERK’ as well as ‘phosphorylation of ERK’ (30). Through this fine-tuning of magnitude and duration of ERK activation, the ERK signal might have selectivity for suitable targets from a large kinase network that regulates a variety of physiological processes. Supporting this idea, our statistical analysis indicated the poor prognostic impact of DUSP6/MKP3, and these findings suggested that DUSP6/MKP3 could contribute to ERK activation by restoring the capacity for repeated phosphorylation. Additional evidence of the positive action of DUSP6/MKP3 was provided by the correlation between the expression levels of ERK and DUSP6/MKP3. Moreover, the FRO cells treated with SiDUSP6 showed decreased cell viability and migration rates compared with SiRNA control. These in vitro experiments consistently supported the positive actions of DUSP6/MKP3.

In conclusion, we confirmed that phospho-ERK1/2 is barely detectable in PTC by IHC staining with two antibodies against phospho-p44/42 MAPK derived from different clones. Furthermore, nuclear localization of ERK was not a hallmark of BRAFV600E-positive PTC. These data suggested that phosphorylation and nuclear localization of ERK is not a predictor of proliferating activity in BRAFV600E-positive PTC. Interestingly, we observed that expression of DUSP6/MKP3 was markedly increased in PTC, and expression of c-Fos and PCNA was higher in BRAFV600E-positive PTC than in BRAFV600E-negative PTC. Furthermore, the link between higher DUSP6 expression and high-risk features of PTC suggests that DUSP6/MKP3 is an important independent factor affecting the signaling pathways in established PTC. Although we could not observe an association between BRAFV600E and DUSP6/MKP3 expressions, the precise role of aberrant DUSP6/MKP3 expression in PTC should be investigated further to better understand the significance of BRAFV600E-positive PTC.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/EJE-12-0010.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported in part by the second phase of the Brain Korea 21 program of the Ministry of Education and by the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A100588). M H Lee, S E Lee, and Y S Jo were supported by NRF/MEST (No. 2010-0005462).

Author contribution statement

J U Lee, S Huang, M H Lee, and S E Lee carried out the immunohistochemical analysis and in vitro experiments and participated in drafting the manuscript. M J Ryu, S J Kim, Y K Kim, S Y K, and K H Joung participated in the design of the study and performed statistical analyses. J M Kim, M Shong, and Y S Jo conceived the study and participated in its progression and coordination. All authors read and approved the final manuscript.

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*

(J U Lee, S Huang and M H Lee contributed equally to this work)

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    Representative figures of immunohistochemical staining for phospho-ERK1/2, ERK1/2, and DUSP6. (A) In normal follicular cells, phospho-ERK1/2 is mainly detected in the nucleus. (B) Phospho-ERK1/2 is undetectable in tumor cells, but surrounding normal follicular cells and stromal cells show high staining intensity of phospho-ERK1/2. (C and D) Tumor cells show markedly increased DUSP6 expression. DUSP6 is exclusively located in the cytoplasm. (E and F) Phospho-ERK1/2-positive cases show focal staining intensity in limited regions. (G) In these regions (E and F), DUSP6 expression is notably decreased. (H and I) Another case showing focal phospho-ERK staining intensity (H) arrows indicate decreased DUSP6 expression (I) in the phospho-ERK1/2 detectable area.

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    DUSP6 silencing affected tumor cell migration. (A) The siDUSP6 treatment markedly decreased the DUSP6 expression compared with the siCTL treatment of the FRO cells. (B) The MTT assay demonstrating the cell viability of the siDUSP6- and siCTL-treated FRO cells. (C and D) The scratch assays were performed using the siDUSP6- and siCTL-treated FRO cells. Average means were compared with Mann–Whitney U test. All the reported P values are two sided. *P<0.05; **P<0.01. The data represent the mean±s.d. of three independent experiments.