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Phosphorylated c-Jun NH2-terminal kinase is overexpressed in human papillary thyroid carcinomas and associates with lymph node metastasis
Cancer Letters 293 (2010) 175–180
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Phosphorylated c-Jun NH2-terminal kinase is overexpressed in human papillary thyroid carcinomas and associates with lymph node metastasis Xiao Wang a,*,1, Lan Chao b,1, Junhui Zhen c, Liansheng Chen a, Guohui Ma a, Xin Li a a
Department of Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan 250012, China Infertility Center, Qilu Hospital, Shandong University, Jinan 250012, China c Department of Pathology, Shandong University School of Medicine, Jinan 250012, China b
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 27 December 2009 Accepted 11 January 2010
Keywords: Papillary thyroid carcinoma Immunohistochemistry Western blot p-JNK Lymph node metastasis
a b s t r a c t To evaluate the associations of phosphorylated c-Jun NH2-terminal kinase (p-JNK) expression with clinicopathological features in patients with papillary thyroid carcinoma, p-JNK expression were immunohistochemically measured in 121 thyroid samples. p-JNK was overexpressed in papillary thyroid carcinomas with respect to matched nontumorous tissues (P = 0.000), which was supported by western blot analysis. Increased p-JNK expression was significantly associated with the presence of lymph node metastases (P = 0.001) and advanced TNM stages (P = 0.02). Furthermore, p-JNK expression was positively correlated with osteopontin (OPN) expression (r = 0.58, P < 0.001). Activation of p-JNK may play a role in the carcinogenesis and lymph node metastasis of papillary thyroid carcinoma, and may be a molecular target for therapeutic intervention. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Papillary thyroid carcinomas far outnumber the other morphological subtypes [1]. Hallmarks of papillary thyroid carcinoma are chromosomal translocations or inversions that cause the recombination of the tyrosine kinase domain of the RET receptor to heterologous genes, thereby generating RET/PTC chimeric oncogenes [2]. A remarkable feature of this malignancy is the high frequency of lymph node metastasis. The presence of lymph node metastasis is associated with a greater risk of recurrent disease [3]. The high frequency of lymph node metastasis in papillary thyroid carcinoma contrasts with the low frequency of hematogenous dissemination, suggesting preferential metastatic spread via the lymphatic pathway. In the new tumor, node, metastases (TNM) staging system, lymph node metastasis is a more important factor than tumor size
* Corresponding author. Tel.: +86 531 85695023; fax: +86 531 86117071. E-mail address: [email protected] (X. Wang). 1 Equal contribution. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.01.007
in overall staging [4]. Better understanding of the molecular pathogenesis of papillary thyroid carcinoma is needed to discover thyroid cancer biomarkers, especially those reflecting disease aggressiveness and prognosis [5]. Nevertheless, until now, the factors regulating the metastatic process in papillary thyroid carcinoma remain poorly understood. Mitogen-activated protein kinase (MAPK) are well-conserved enzymes connecting cell-surface receptors to intracellular regulating targets. There are three well-known MAPK subfamilies: extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38 MAPK isoforms [6]. JNK is characterized as a stress activated protein kinase (SAPK). Phosphorylation of JNK (p-JNK) is involved in the BRCA1-facilitated apoptosis in cells derived from osteosarcoma, breast cancer, and ovarian cancer [7,8]. Previous reports indicated that an imperfect association existed between the increased immunoreactivity of activated c-Jun, a downstream effecter of p-JNK, and the poorer survival in breast cancer [9], and the decreased p-JNK1/2 expression was associated with a better overall survival of breast infiltrating ductal carcinoma [10]. Given
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the success of protein kinase inhibitors [11,12], much interest is therefore invested into the development of MAPK inhibitors, in particular JNK inhibitors. Several in vitro experiments demonstrated high basal activation of JNK in thyroid cell lines [13–15]. Furthermore, Yang and his colleagues reported that p-JNK expression was increased in papillary thyroid tumors compared with paired normal tissues [16]. However, the clinical information about the role of p-JNK in papillary thyroid carcinoma is lacking. The objective of this study was to evaluate the expression of p-JNK in patients suffering from papillary thyroid carcinoma by means of immunohistochemistry and Western Blot analyses, and to determine its correlation with clinicopathological features. Furthermore, papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes [5]. Therefore, the association of p-JNK expression with osteopontin (OPN) expression, which is a major RET/PTC oncogene-induced transcriptional target in thyroid cells, was also evaluated.
2. Materials and methods 2.1. Patients and tissue samples Archival thyroid samples from 121 patients who had undergone surgical resection of the thyroid gland for benign or malignant lesions at Jinan Central Hospital of Shandong University (Jinan, China) from May 1996 to March 2007 were retrieved. All these patients, included in this study, signed a written informed consent approved by the ethical committee of the hospital before they underwent surgery, and the study was approved by the institutional review board committee. Those 101 patients with papillary thyroid carcinoma consisted of 77 females and 24 males, and the mean patient age at surgery was 52 years (range, 29–72 years). Total or near total thyroidectomy was performed for 67 patients (66.3%), whereas the remaining patients underwent more limited thyroidectomy such as subtotal thyroidectomy and lobectomy with isthmectomy. Lymph node dissection (central compartment or comprehensive) was performed only in those with ultrasonographic evident of macroscopic adenopathy. Presence of metastasis was defined as patients with macroscopic pathologically confirmed metastasis. Clinicopathological features include tumor size, focality, histology type, lymph node metastasis, hyperplasia, capsule invasion, extrathyroid extension, vascular invasion, and TNM stages were recorded. The TNM staging was classified using the American Joint Committee on Cancer (AJCC) staging system (4). Papillary thyroid carcinomas were subdivided into the following categories according to World Health Organization (WHO) histological classification of thyroid tumors: classic variant, follicular variant, and other variant (including diffuse sclerosing, tall cell variant, and others). Tumor samples and 20 matched nontumorous tissues were obtained immediately after thyroidectomy. In addition, 10 follicular adenomas, and 10 multinodular goiters were examined. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin blocks.
Furthermore, 17 fresh paired samples of primary papillary thyroid carcinoma and the corresponding nontumorous tissues were obtained from the same patients, snap frozen in liquid nitrogen, and kept at 80 °C until further analysis. 2.2. Immunohistochemical staining Phosphorylated JNK and OPN expressions were determined by immunohistochemical staining of tissue sections using affinity-purified rabbit anti-phospho-JNK antibody (AF1205, T183/Y185, 1:100 dilution. R&D Systems, Minneapolis, MN) and mouse anti-human monoclonal osteopontin antibody (MAb1433, 1:200 dilution. R&D Systems, Minneapolis, MN). Histopathological assessment was performed on paraffin sections of 5 lm in thickness. Immunohistochemistry was performed on sections after incubating the tissue in 3% aqueous hydrogen peroxide for 10 min, followed by washing in 0.01 mmol/L phosphate-buffered saline and treatment with 1% normal goat serum for 10 min at room temperature. Antigen retrieval was done by heating sections in 0.01 mol/L sodium citrate buffer (pH 6.0) using a microwave oven (620 W) for three 5-min periods. Sites of primary antibody binding were identified by incubating blots with horseradish peroxidase-conjugated anti-rabbit/ mouse IgG (Santa Cruz Biotechnology, dilution 1:250. Santa Cruz, CA) followed by reaction with substrate, 3,30 diaminobenzidine tetrahydrochloride (DAB). A section of normal human breast, previously proven to be p-JNK positive was used as a positive control. Normal gallbladder was used as positive control for OPN. Omission of the primary antibody was used as negative control. 2.3. Evaluation of immunohistochemical staining Immunohistochemical staining was evaluated and scored by two pathologists (L.C., J.Z.) who worked independently and were blinded to clinical information. All the pathological images were evaluated during 1 month and agreement on the staining scores was by consensus. The expression of p-JNK was cytoplasmic and/or nuclear, whereas the OPN immunostaining was mostly localized in cytoplasm. The overall amount of staining for p-JNK and OPN were determined based on staining intensity (I) and proportion/extent (E) of stained tumor cells. A proportion score was assigned to represent the estimated proportion of positively stained tumor cells (0 = none, 1 6 1/100, 2 = 1/100–1/10, 3 = 1/10–1/3, 4 = 1/3–2/3, 5 P 2/3). The average estimated intensity of staining in positive cells was assigned as an intensity score (0 = none, 1 = weak, 2 = intermediate, 3 = strong). The proportion score and intensity score were added to obtain a total score ranging from 0 to 8 (0 for negative staining and 2–8 for positive staining ranges). 2.4. Western blot analysis Total protein lysates were made from frozen tissues using RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were measured using the
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BCA™ Protein Assay Kit (Pierce, Rockford, IL). 30 lg of proteins in every sample were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and the objective proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P. Millipore Corp., Bedford, MA). The membranes were washed with Tris-buffered saline – Tween-20 (TBS-T) (0.2 M NaCl, 25 mM Tris, pH 7.5, 0.5 mL/L Tween-20) three times (5 min once). After blocking in TBS-T with 5% non-fat dry milk or bovine serum albumin (BSA) overnight at 4 °C, membranes were incubated with anti-p-JNK polyclonal antibody (AF1205, T183/Y185, 1:1000 dilution. R&D Systems, Minneapolis, MN), anti-OPN polyclonal primary antibody (MAb1433, 1:1000 dilution. R&D Systems, Minneapolis, MN), anti-JNK polyclonal antibody (9252, 1:1000 dilution. Cell Signaling Technology, Beverly, MA), and monoclonal anti-b actin antibody (1:5000 dilution. R&D Systems, Minneapolis, MN) for 2 h at room temperature. The membrane was washed three times with TBS-T for 5 min each, incubated for 1 h in TBS-T with HRP conjugated goat anti-rabbit/mouse secondary antibody (Santa Cruz Biotechnology, dilution 1:2000–5000. Santa Cruz, CA), and washed five times with TBS-T for 5 min each. Antibodies bound to membrane immobilized proteins were visualized by enhanced chemiluminescence using
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the ECL TM western blotting detection reagents (GE Healthcare, Piscataway, NJ). 2.5. Statistical analysis Frequency tables were analyzed using the v2 test and Spearman’s rank (r) correlation coefficient. All statistical tests were 2-sided. A statistical significance of observed difference was set at P < 0.05. All data were analyzed using SPSS statistical software (Version 13.0, SPSS Inc., Chicago, IL). 3. Results 3.1. Clinicopathological parameters of the patients Papillary thyroid carcinomas were subdivided into the following categories according to WHO histological classification of thyroid tumors: 57 classic variants, 30 follicular variants, and 14 other variants (six tall cell variants, four diffuse selerosing variants, three Warthin-like tumors, and one cribriform morular variant). Clinical staging results were: stage I, 34 patients (33.7%); stage II, 30 patients (29.7%); stage III, 28 patients (27.7%); and stage IV, nine patients (8.9%). Fifty-eight out of 101 patients had lymph node metastases. Overall, p-JNK was detected (either cytoplasmic or nuclear staining) in 60 (59.4%) of the 101 papillary thyroid carcinomas, 3 (15%) of 20 matched nontumorous tissues, 2 (20%) of 10 multinodular goiters, 2 (20%) of 10 follicular adenomas (Fig. 1A–D). Thus,
Fig. 1. p-JNK expression in benign and malignant tissues of thyroid gland: (A) (magnification. 100) Negative expression of p-JNK in a matched nontumorous thyroid tissue (p-JNK score = 1). (B) (magnification. 100) Cytoplastic staining of p-JNK protein in a nodular goiter tissue (p-JNK score = 4). (C) (magnification. 200) Cytoplastic expression of p-JNK in a follicular adenoma tissue (p-JNK score = 7). (D) (magnification. 200) Strong expression of p-JNK in a papillary thyroid carcinoma tissue (p-JNK score = 8).
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Table 1 Correlations between p-JNK expression and clinicopathological parameters in papillary thyroid carcinoma. Characteristics
n
p-JNK expression (%)
p-value
Positive (n = 60)
Negative (n = 41)
Age 645 years >45 years
50 51
28 (46.7%) 32 (53.3%)
22 (53.7%) 19 (46.3%)
0.63
Gender Male Female
77 24
45 (75%) 15 (25%)
32 (78%) 9 (22%)
0.91
Histology Classic variant Follicular variant Other variant
57 30 14
34 (56.7%) 17 (28.3%) 9 (15.0%)
23 (56.1%) 13 (31.7%) 5 (12.2%)
0.89
Stages I/II III/IV
64 37
32 (53.3%) 28 (36.7%)
32 (78.1%) 9 (21.9%)
0.02
Focality Unifocal Multifocal
55 36
31 (51.7%) 29 (48.3%)
24 (58.5%) 17 (41.5%)
0.63
Lymph node metastasis Yes No
58 43
43 (71.7%) 17 (28.3%)
15 (36.6%) 26 (63.4%)
0.001
Tumor size 62 cm >2 cm
52 49
31 (51.7%) 29 (48.3%)
21 (51.2%) 20 (48.8%)
0.97
Hyperplasia Yes No
37 64
25 (41.7%) 35 (58.3%)
12 (29.3%) 29 (70.7%)
0.29
Capsule invasion Yes No
42 59
29 (48.3%) 31 (51.7%)
13 (31.7%) 28 (68.3%)
0.14
Extrathyroid extension Yes No
24 77
13 (21.7%) 47 (78.3%)
11 (26.8%) 30 (73.2%)
0.72
Vascular invasion Yes No
27 74
19 (31.7%) 41 (68.3%)
8 (19.5%) 33 (80.5%)
0.26
OPN Positive Negative
59 42
42 (70.0%) 18 (30.0%)
17 (41.5%) 24 (58.5%)
0.008
p-JNK was overexpressed in papillary thyroid carcinomas with respect to matched nontumorous tissues (P = 0.000), follicular adenomas (P = 0.017), and multinodular goiters (P = 0.017).
was activated together. There was a highly significant positive association between p-JNK expression and OPN expression in papillary thyroid carcinomas (r = 0.58, P < 0.001) (Table 2).
3.2. Associations of p-JNK expression with clinicopathological parameters
3.4. Western blot analysis of protein expression
Of the 101 papillary thyroid carcinomas, p-JNK expression was significantly correlated with increased TNM stages. Positive expression of p-JNK was more common in patients with III/IV stages compared to those with pJNK negative expression (36.7% vs. 21.9%, P = 0.02) (Table 1). Lymph node metastases were more common in cancers with p-JNK positive expression compared to those with p-JNK negative expression (71.7% vs. 36.6%, P = 0.001). Furthermore, tumors with p-JNK positive expression frequently more showed OPN positivity when compared to those with p-JNK negative expression (70.0% vs. 41.5%, P = 0.008). There was no significant difference in ages, gender, histology type, focality, tumor size, hyperplasia, capsule invasion, extrathyroid extension, and vascular invasion between the carcinomas with p-JNK positive or negative expression (P > 0.05) (Table 1).
To confirm the overexpression of p-JNK and OPN in epithelial thyroid carcinomas at protein level, Western blot analyses were performed using protein extracts obtained from 17 papillary thyroid carcinoma samples and corresponding nontumorous tissues. Representative results of Western blot analysis are showed in Fig. 2. The relative intensity of each reactive p-JNK, total JNK, or OPN band were normalized to b-actin, used as loading control. The values obtained from the tumors were in turn expressed relative to the levels detected in the corresponding nontumorous thyroid tissues. We concluded that 13 out of the 17 tumors (76.5%) expressed p-JNK protein at levels higher than that of p-JNK protein in the corresponding thyroid tissue, and nine cases (52.9%) expressed higher total JNK protein compared with corresponding nontumorous tissue. 64.7% (11 of 17) of papillary thyroid carcinoma samples were found to have a higher expression of OPN protein in the tumors compared with their corresponding nontumorous tissues. This analysis is in agreement with the immunohistochemical studies, indicating that a high proportion of papillary thyroid carcinomas expressed significantly increased levels of p-JNK
3.3. Association of p-JNK expression with OPN expression OPN was detected in 59 (58.4%) of the 101 papillary thyroid carcinomas (Table 1). In 47 (47.5%) of the 101 tissues, p-JNK and OPN expression
X. Wang et al. / Cancer Letters 293 (2010) 175–180 Table 2 Correlations between p-JNK expression and OPN expression. Parameters
r
p-value
p-JNK vs. OPN
0.58
<0.001
Fig. 2. Representative results of Western blot analysis showing protein expression of p-JNK, total JNK, and OPN in papillary thyroid carcinoma (T) and corresponding nontumorous tissues (N). Actin is shown to demonstrate the same amount of proteins located on the gel.
or OPN protein. Importantly, the western blot analysis also demonstrates that p-JNK expression was positively correlated with OPN expression. In 12 (70.6%) of the 17 tumors with papillary thyroid carcinoma, p-JNK and OPN expression was activated or reduced together. Thus, western analysis of these proteins in epithelial thyroid carcinoma tissues confirms the immunoreactivity data described above.
4. Discussion It was reported that activation of JNK was important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity was necessary to promote proliferation and maintain diploidy in breast cancer cells [17]. Furthermore, Davidson et al. believed that the elevated JNK activation in effusions may be a stress-related mechanism providing breast carcinoma cells with survival advantages rather than a drive towards apoptosis [18]. In the present study, p-JNK was overexpressed in human papillary thyroid carcinomas with respect to matched nontumorous tissues in the immunohistochemical analysis, which was supported by Western blot analysis, suggesting that phosphorylation of JNK play a role in the carcinogenesis of papillary thyroid carcinoma. Shimada et al. demonstrated that JNK activation could account for highly invasive phenotypes of urothelial carcinoma cells and contribute to cancer development through MMP-9 and VEGF induction in vitro and in vivo [19]. In this study we have shown that p-JNK positive expression correlate with aggressive clinicopathological features of papillary thyroid carcinoma. Indeed, the presence of lymph node metastases and advanced TNM stages both positively correlated with p-JNK positivity. Furthermore, tumors with p-JNK positive expression frequently more showed OPN positivity, which was regarded as a key molecular player involved in lymphatic metastasis of breast cancer [20]. Therefore, activated JNK may play a role not only in carcinogenesis but also in lymph node metastasis of papillary thyroid carcinoma. OPN, an acidic hydrophilic glycophosphoprotein, is the major phosphoprotein secreted by malignant cells in patients with advanced metastatic
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cancer, and has been implicated in tumor cell migration and metastasis. Data suggest that OPN overexpression represents a key molecular event in tumor progression and metastasis [21–26]. Selkirk et al. reported that the enhancement of JNK1 negative feedback on OPN stimulated cellular processes may therefore represented an important therapeutic target for human central nervous system (CNS) malignancy [27]. Furthermore, Kuo and his colleagues indicated that constitutive JNK activation in mammary epithelial tumor 4T1 cells phosphorylated cJun to ultimately promote OPN transcription in 4T1 cells [28]. Coordinated expression of p-JNK and OPN immunoreaction, confirmed also by western blot analysis, was detected in 47.5% of papillary thyroid carcinoma lesions in the current study, suggesting an involvement of both genes in the same molecular pathway in a subgroup of lesions. Currently available chemotherapy is ineffective against thyroid cancer. Therefore, there is critical need to develop new therapies directed against pathways that are activated in these tumors. The MAPK kinase pathways could be targets for thyroid cancer therapy [29]. Khatlani et al. concluded that JNK activation promoted lung tumorigenesis in a subset of patients, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of non-small cell lung cancer (NSCLC) [30]. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of hepatocellular carcinoma (HCC) cells via attenuation of paxillin phosphorylation at Ser178 [31]. Several peptide inhibitors have been developed, such as JNKI1 [32], which had been successfully used in mouse models for HCC [33]. BI-78D3, which inhibits JNK activity through interfering with binding to the JNK-interacting protein 1 (JiP1) scaffold, represents not only a JNK inhibitor but also a promising stepping stone toward the development of an innovative class of therapeutics [34]. As the field of JNK inhibitors is rapidly moving, it is anticipated that several targeted therapies with new drugs will be successfully applied and used in the clinic in the near future. To better select subsets of patients who would benefit from such approaches will require a better understanding of JNK’s role in the context of specific genetic mutations. Given the associations of positive p-JNK expression with aggressive clinicopathological features in the current study, the p-JNK may have the potential value as an attractive target for therapeutic management in patients with papillary thyroid carcinoma. To our knowledge, this is the first study to demonstrate the association between p-JNK expression and clinicopathological features in the patients with papillary thyroid carcinoma. Activation of the JNK signaling pathway appears to be an important event in thyroid tumorigenesis and, perhaps, in tumor progression making p-JNK an attractive target for pharmaceutical development. Conflict of interest None declared. Reference [1] S.I. Sherman, Thyroid carcinoma, Lancet 361 (2003) 501–511.
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Phosphorylated c-Jun NH2-terminal kinase is overexpressed in human papillary thyroid carcinomas and associates with lymph node metastasis Xiao Wang a,*,1, Lan Chao b,1, Junhui Zhen c, Liansheng Chen a, Guohui Ma a, Xin Li a a
Department of Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan 250012, China Infertility Center, Qilu Hospital, Shandong University, Jinan 250012, China c Department of Pathology, Shandong University School of Medicine, Jinan 250012, China b
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 27 December 2009 Accepted 11 January 2010
Keywords: Papillary thyroid carcinoma Immunohistochemistry Western blot p-JNK Lymph node metastasis
a b s t r a c t To evaluate the associations of phosphorylated c-Jun NH2-terminal kinase (p-JNK) expression with clinicopathological features in patients with papillary thyroid carcinoma, p-JNK expression were immunohistochemically measured in 121 thyroid samples. p-JNK was overexpressed in papillary thyroid carcinomas with respect to matched nontumorous tissues (P = 0.000), which was supported by western blot analysis. Increased p-JNK expression was significantly associated with the presence of lymph node metastases (P = 0.001) and advanced TNM stages (P = 0.02). Furthermore, p-JNK expression was positively correlated with osteopontin (OPN) expression (r = 0.58, P < 0.001). Activation of p-JNK may play a role in the carcinogenesis and lymph node metastasis of papillary thyroid carcinoma, and may be a molecular target for therapeutic intervention. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Papillary thyroid carcinomas far outnumber the other morphological subtypes [1]. Hallmarks of papillary thyroid carcinoma are chromosomal translocations or inversions that cause the recombination of the tyrosine kinase domain of the RET receptor to heterologous genes, thereby generating RET/PTC chimeric oncogenes [2]. A remarkable feature of this malignancy is the high frequency of lymph node metastasis. The presence of lymph node metastasis is associated with a greater risk of recurrent disease [3]. The high frequency of lymph node metastasis in papillary thyroid carcinoma contrasts with the low frequency of hematogenous dissemination, suggesting preferential metastatic spread via the lymphatic pathway. In the new tumor, node, metastases (TNM) staging system, lymph node metastasis is a more important factor than tumor size
* Corresponding author. Tel.: +86 531 85695023; fax: +86 531 86117071. E-mail address: [email protected] (X. Wang). 1 Equal contribution. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.01.007
in overall staging [4]. Better understanding of the molecular pathogenesis of papillary thyroid carcinoma is needed to discover thyroid cancer biomarkers, especially those reflecting disease aggressiveness and prognosis [5]. Nevertheless, until now, the factors regulating the metastatic process in papillary thyroid carcinoma remain poorly understood. Mitogen-activated protein kinase (MAPK) are well-conserved enzymes connecting cell-surface receptors to intracellular regulating targets. There are three well-known MAPK subfamilies: extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38 MAPK isoforms [6]. JNK is characterized as a stress activated protein kinase (SAPK). Phosphorylation of JNK (p-JNK) is involved in the BRCA1-facilitated apoptosis in cells derived from osteosarcoma, breast cancer, and ovarian cancer [7,8]. Previous reports indicated that an imperfect association existed between the increased immunoreactivity of activated c-Jun, a downstream effecter of p-JNK, and the poorer survival in breast cancer [9], and the decreased p-JNK1/2 expression was associated with a better overall survival of breast infiltrating ductal carcinoma [10]. Given
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the success of protein kinase inhibitors [11,12], much interest is therefore invested into the development of MAPK inhibitors, in particular JNK inhibitors. Several in vitro experiments demonstrated high basal activation of JNK in thyroid cell lines [13–15]. Furthermore, Yang and his colleagues reported that p-JNK expression was increased in papillary thyroid tumors compared with paired normal tissues [16]. However, the clinical information about the role of p-JNK in papillary thyroid carcinoma is lacking. The objective of this study was to evaluate the expression of p-JNK in patients suffering from papillary thyroid carcinoma by means of immunohistochemistry and Western Blot analyses, and to determine its correlation with clinicopathological features. Furthermore, papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes [5]. Therefore, the association of p-JNK expression with osteopontin (OPN) expression, which is a major RET/PTC oncogene-induced transcriptional target in thyroid cells, was also evaluated.
2. Materials and methods 2.1. Patients and tissue samples Archival thyroid samples from 121 patients who had undergone surgical resection of the thyroid gland for benign or malignant lesions at Jinan Central Hospital of Shandong University (Jinan, China) from May 1996 to March 2007 were retrieved. All these patients, included in this study, signed a written informed consent approved by the ethical committee of the hospital before they underwent surgery, and the study was approved by the institutional review board committee. Those 101 patients with papillary thyroid carcinoma consisted of 77 females and 24 males, and the mean patient age at surgery was 52 years (range, 29–72 years). Total or near total thyroidectomy was performed for 67 patients (66.3%), whereas the remaining patients underwent more limited thyroidectomy such as subtotal thyroidectomy and lobectomy with isthmectomy. Lymph node dissection (central compartment or comprehensive) was performed only in those with ultrasonographic evident of macroscopic adenopathy. Presence of metastasis was defined as patients with macroscopic pathologically confirmed metastasis. Clinicopathological features include tumor size, focality, histology type, lymph node metastasis, hyperplasia, capsule invasion, extrathyroid extension, vascular invasion, and TNM stages were recorded. The TNM staging was classified using the American Joint Committee on Cancer (AJCC) staging system (4). Papillary thyroid carcinomas were subdivided into the following categories according to World Health Organization (WHO) histological classification of thyroid tumors: classic variant, follicular variant, and other variant (including diffuse sclerosing, tall cell variant, and others). Tumor samples and 20 matched nontumorous tissues were obtained immediately after thyroidectomy. In addition, 10 follicular adenomas, and 10 multinodular goiters were examined. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin blocks.
Furthermore, 17 fresh paired samples of primary papillary thyroid carcinoma and the corresponding nontumorous tissues were obtained from the same patients, snap frozen in liquid nitrogen, and kept at 80 °C until further analysis. 2.2. Immunohistochemical staining Phosphorylated JNK and OPN expressions were determined by immunohistochemical staining of tissue sections using affinity-purified rabbit anti-phospho-JNK antibody (AF1205, T183/Y185, 1:100 dilution. R&D Systems, Minneapolis, MN) and mouse anti-human monoclonal osteopontin antibody (MAb1433, 1:200 dilution. R&D Systems, Minneapolis, MN). Histopathological assessment was performed on paraffin sections of 5 lm in thickness. Immunohistochemistry was performed on sections after incubating the tissue in 3% aqueous hydrogen peroxide for 10 min, followed by washing in 0.01 mmol/L phosphate-buffered saline and treatment with 1% normal goat serum for 10 min at room temperature. Antigen retrieval was done by heating sections in 0.01 mol/L sodium citrate buffer (pH 6.0) using a microwave oven (620 W) for three 5-min periods. Sites of primary antibody binding were identified by incubating blots with horseradish peroxidase-conjugated anti-rabbit/ mouse IgG (Santa Cruz Biotechnology, dilution 1:250. Santa Cruz, CA) followed by reaction with substrate, 3,30 diaminobenzidine tetrahydrochloride (DAB). A section of normal human breast, previously proven to be p-JNK positive was used as a positive control. Normal gallbladder was used as positive control for OPN. Omission of the primary antibody was used as negative control. 2.3. Evaluation of immunohistochemical staining Immunohistochemical staining was evaluated and scored by two pathologists (L.C., J.Z.) who worked independently and were blinded to clinical information. All the pathological images were evaluated during 1 month and agreement on the staining scores was by consensus. The expression of p-JNK was cytoplasmic and/or nuclear, whereas the OPN immunostaining was mostly localized in cytoplasm. The overall amount of staining for p-JNK and OPN were determined based on staining intensity (I) and proportion/extent (E) of stained tumor cells. A proportion score was assigned to represent the estimated proportion of positively stained tumor cells (0 = none, 1 6 1/100, 2 = 1/100–1/10, 3 = 1/10–1/3, 4 = 1/3–2/3, 5 P 2/3). The average estimated intensity of staining in positive cells was assigned as an intensity score (0 = none, 1 = weak, 2 = intermediate, 3 = strong). The proportion score and intensity score were added to obtain a total score ranging from 0 to 8 (0 for negative staining and 2–8 for positive staining ranges). 2.4. Western blot analysis Total protein lysates were made from frozen tissues using RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were measured using the
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BCA™ Protein Assay Kit (Pierce, Rockford, IL). 30 lg of proteins in every sample were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and the objective proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P. Millipore Corp., Bedford, MA). The membranes were washed with Tris-buffered saline – Tween-20 (TBS-T) (0.2 M NaCl, 25 mM Tris, pH 7.5, 0.5 mL/L Tween-20) three times (5 min once). After blocking in TBS-T with 5% non-fat dry milk or bovine serum albumin (BSA) overnight at 4 °C, membranes were incubated with anti-p-JNK polyclonal antibody (AF1205, T183/Y185, 1:1000 dilution. R&D Systems, Minneapolis, MN), anti-OPN polyclonal primary antibody (MAb1433, 1:1000 dilution. R&D Systems, Minneapolis, MN), anti-JNK polyclonal antibody (9252, 1:1000 dilution. Cell Signaling Technology, Beverly, MA), and monoclonal anti-b actin antibody (1:5000 dilution. R&D Systems, Minneapolis, MN) for 2 h at room temperature. The membrane was washed three times with TBS-T for 5 min each, incubated for 1 h in TBS-T with HRP conjugated goat anti-rabbit/mouse secondary antibody (Santa Cruz Biotechnology, dilution 1:2000–5000. Santa Cruz, CA), and washed five times with TBS-T for 5 min each. Antibodies bound to membrane immobilized proteins were visualized by enhanced chemiluminescence using
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the ECL TM western blotting detection reagents (GE Healthcare, Piscataway, NJ). 2.5. Statistical analysis Frequency tables were analyzed using the v2 test and Spearman’s rank (r) correlation coefficient. All statistical tests were 2-sided. A statistical significance of observed difference was set at P < 0.05. All data were analyzed using SPSS statistical software (Version 13.0, SPSS Inc., Chicago, IL). 3. Results 3.1. Clinicopathological parameters of the patients Papillary thyroid carcinomas were subdivided into the following categories according to WHO histological classification of thyroid tumors: 57 classic variants, 30 follicular variants, and 14 other variants (six tall cell variants, four diffuse selerosing variants, three Warthin-like tumors, and one cribriform morular variant). Clinical staging results were: stage I, 34 patients (33.7%); stage II, 30 patients (29.7%); stage III, 28 patients (27.7%); and stage IV, nine patients (8.9%). Fifty-eight out of 101 patients had lymph node metastases. Overall, p-JNK was detected (either cytoplasmic or nuclear staining) in 60 (59.4%) of the 101 papillary thyroid carcinomas, 3 (15%) of 20 matched nontumorous tissues, 2 (20%) of 10 multinodular goiters, 2 (20%) of 10 follicular adenomas (Fig. 1A–D). Thus,
Fig. 1. p-JNK expression in benign and malignant tissues of thyroid gland: (A) (magnification. 100) Negative expression of p-JNK in a matched nontumorous thyroid tissue (p-JNK score = 1). (B) (magnification. 100) Cytoplastic staining of p-JNK protein in a nodular goiter tissue (p-JNK score = 4). (C) (magnification. 200) Cytoplastic expression of p-JNK in a follicular adenoma tissue (p-JNK score = 7). (D) (magnification. 200) Strong expression of p-JNK in a papillary thyroid carcinoma tissue (p-JNK score = 8).
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Table 1 Correlations between p-JNK expression and clinicopathological parameters in papillary thyroid carcinoma. Characteristics
n
p-JNK expression (%)
p-value
Positive (n = 60)
Negative (n = 41)
Age 645 years >45 years
50 51
28 (46.7%) 32 (53.3%)
22 (53.7%) 19 (46.3%)
0.63
Gender Male Female
77 24
45 (75%) 15 (25%)
32 (78%) 9 (22%)
0.91
Histology Classic variant Follicular variant Other variant
57 30 14
34 (56.7%) 17 (28.3%) 9 (15.0%)
23 (56.1%) 13 (31.7%) 5 (12.2%)
0.89
Stages I/II III/IV
64 37
32 (53.3%) 28 (36.7%)
32 (78.1%) 9 (21.9%)
0.02
Focality Unifocal Multifocal
55 36
31 (51.7%) 29 (48.3%)
24 (58.5%) 17 (41.5%)
0.63
Lymph node metastasis Yes No
58 43
43 (71.7%) 17 (28.3%)
15 (36.6%) 26 (63.4%)
0.001
Tumor size 62 cm >2 cm
52 49
31 (51.7%) 29 (48.3%)
21 (51.2%) 20 (48.8%)
0.97
Hyperplasia Yes No
37 64
25 (41.7%) 35 (58.3%)
12 (29.3%) 29 (70.7%)
0.29
Capsule invasion Yes No
42 59
29 (48.3%) 31 (51.7%)
13 (31.7%) 28 (68.3%)
0.14
Extrathyroid extension Yes No
24 77
13 (21.7%) 47 (78.3%)
11 (26.8%) 30 (73.2%)
0.72
Vascular invasion Yes No
27 74
19 (31.7%) 41 (68.3%)
8 (19.5%) 33 (80.5%)
0.26
OPN Positive Negative
59 42
42 (70.0%) 18 (30.0%)
17 (41.5%) 24 (58.5%)
0.008
p-JNK was overexpressed in papillary thyroid carcinomas with respect to matched nontumorous tissues (P = 0.000), follicular adenomas (P = 0.017), and multinodular goiters (P = 0.017).
was activated together. There was a highly significant positive association between p-JNK expression and OPN expression in papillary thyroid carcinomas (r = 0.58, P < 0.001) (Table 2).
3.2. Associations of p-JNK expression with clinicopathological parameters
3.4. Western blot analysis of protein expression
Of the 101 papillary thyroid carcinomas, p-JNK expression was significantly correlated with increased TNM stages. Positive expression of p-JNK was more common in patients with III/IV stages compared to those with pJNK negative expression (36.7% vs. 21.9%, P = 0.02) (Table 1). Lymph node metastases were more common in cancers with p-JNK positive expression compared to those with p-JNK negative expression (71.7% vs. 36.6%, P = 0.001). Furthermore, tumors with p-JNK positive expression frequently more showed OPN positivity when compared to those with p-JNK negative expression (70.0% vs. 41.5%, P = 0.008). There was no significant difference in ages, gender, histology type, focality, tumor size, hyperplasia, capsule invasion, extrathyroid extension, and vascular invasion between the carcinomas with p-JNK positive or negative expression (P > 0.05) (Table 1).
To confirm the overexpression of p-JNK and OPN in epithelial thyroid carcinomas at protein level, Western blot analyses were performed using protein extracts obtained from 17 papillary thyroid carcinoma samples and corresponding nontumorous tissues. Representative results of Western blot analysis are showed in Fig. 2. The relative intensity of each reactive p-JNK, total JNK, or OPN band were normalized to b-actin, used as loading control. The values obtained from the tumors were in turn expressed relative to the levels detected in the corresponding nontumorous thyroid tissues. We concluded that 13 out of the 17 tumors (76.5%) expressed p-JNK protein at levels higher than that of p-JNK protein in the corresponding thyroid tissue, and nine cases (52.9%) expressed higher total JNK protein compared with corresponding nontumorous tissue. 64.7% (11 of 17) of papillary thyroid carcinoma samples were found to have a higher expression of OPN protein in the tumors compared with their corresponding nontumorous tissues. This analysis is in agreement with the immunohistochemical studies, indicating that a high proportion of papillary thyroid carcinomas expressed significantly increased levels of p-JNK
3.3. Association of p-JNK expression with OPN expression OPN was detected in 59 (58.4%) of the 101 papillary thyroid carcinomas (Table 1). In 47 (47.5%) of the 101 tissues, p-JNK and OPN expression
X. Wang et al. / Cancer Letters 293 (2010) 175–180 Table 2 Correlations between p-JNK expression and OPN expression. Parameters
r
p-value
p-JNK vs. OPN
0.58
<0.001
Fig. 2. Representative results of Western blot analysis showing protein expression of p-JNK, total JNK, and OPN in papillary thyroid carcinoma (T) and corresponding nontumorous tissues (N). Actin is shown to demonstrate the same amount of proteins located on the gel.
or OPN protein. Importantly, the western blot analysis also demonstrates that p-JNK expression was positively correlated with OPN expression. In 12 (70.6%) of the 17 tumors with papillary thyroid carcinoma, p-JNK and OPN expression was activated or reduced together. Thus, western analysis of these proteins in epithelial thyroid carcinoma tissues confirms the immunoreactivity data described above.
4. Discussion It was reported that activation of JNK was important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity was necessary to promote proliferation and maintain diploidy in breast cancer cells [17]. Furthermore, Davidson et al. believed that the elevated JNK activation in effusions may be a stress-related mechanism providing breast carcinoma cells with survival advantages rather than a drive towards apoptosis [18]. In the present study, p-JNK was overexpressed in human papillary thyroid carcinomas with respect to matched nontumorous tissues in the immunohistochemical analysis, which was supported by Western blot analysis, suggesting that phosphorylation of JNK play a role in the carcinogenesis of papillary thyroid carcinoma. Shimada et al. demonstrated that JNK activation could account for highly invasive phenotypes of urothelial carcinoma cells and contribute to cancer development through MMP-9 and VEGF induction in vitro and in vivo [19]. In this study we have shown that p-JNK positive expression correlate with aggressive clinicopathological features of papillary thyroid carcinoma. Indeed, the presence of lymph node metastases and advanced TNM stages both positively correlated with p-JNK positivity. Furthermore, tumors with p-JNK positive expression frequently more showed OPN positivity, which was regarded as a key molecular player involved in lymphatic metastasis of breast cancer [20]. Therefore, activated JNK may play a role not only in carcinogenesis but also in lymph node metastasis of papillary thyroid carcinoma. OPN, an acidic hydrophilic glycophosphoprotein, is the major phosphoprotein secreted by malignant cells in patients with advanced metastatic
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cancer, and has been implicated in tumor cell migration and metastasis. Data suggest that OPN overexpression represents a key molecular event in tumor progression and metastasis [21–26]. Selkirk et al. reported that the enhancement of JNK1 negative feedback on OPN stimulated cellular processes may therefore represented an important therapeutic target for human central nervous system (CNS) malignancy [27]. Furthermore, Kuo and his colleagues indicated that constitutive JNK activation in mammary epithelial tumor 4T1 cells phosphorylated cJun to ultimately promote OPN transcription in 4T1 cells [28]. Coordinated expression of p-JNK and OPN immunoreaction, confirmed also by western blot analysis, was detected in 47.5% of papillary thyroid carcinoma lesions in the current study, suggesting an involvement of both genes in the same molecular pathway in a subgroup of lesions. Currently available chemotherapy is ineffective against thyroid cancer. Therefore, there is critical need to develop new therapies directed against pathways that are activated in these tumors. The MAPK kinase pathways could be targets for thyroid cancer therapy [29]. Khatlani et al. concluded that JNK activation promoted lung tumorigenesis in a subset of patients, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of non-small cell lung cancer (NSCLC) [30]. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of hepatocellular carcinoma (HCC) cells via attenuation of paxillin phosphorylation at Ser178 [31]. Several peptide inhibitors have been developed, such as JNKI1 [32], which had been successfully used in mouse models for HCC [33]. BI-78D3, which inhibits JNK activity through interfering with binding to the JNK-interacting protein 1 (JiP1) scaffold, represents not only a JNK inhibitor but also a promising stepping stone toward the development of an innovative class of therapeutics [34]. As the field of JNK inhibitors is rapidly moving, it is anticipated that several targeted therapies with new drugs will be successfully applied and used in the clinic in the near future. To better select subsets of patients who would benefit from such approaches will require a better understanding of JNK’s role in the context of specific genetic mutations. Given the associations of positive p-JNK expression with aggressive clinicopathological features in the current study, the p-JNK may have the potential value as an attractive target for therapeutic management in patients with papillary thyroid carcinoma. To our knowledge, this is the first study to demonstrate the association between p-JNK expression and clinicopathological features in the patients with papillary thyroid carcinoma. Activation of the JNK signaling pathway appears to be an important event in thyroid tumorigenesis and, perhaps, in tumor progression making p-JNK an attractive target for pharmaceutical development. Conflict of interest None declared. Reference [1] S.I. Sherman, Thyroid carcinoma, Lancet 361 (2003) 501–511.
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