In recent years, the incidence of thyroid cancer has increased rapidly,1,2 especially that of the most common malignant form, papillary thyroid cancer (PTC).3
The Hippo pathway was first identified in Drosophila melanogaster during genetic screening to characterize cell growth regulators.4 The Hippo signal transduction pathway is highly conserved in mammals and plays a critical role in tissue and organ size by regulating the balance between cell proliferation and apoptosis.5,6 It consists of a core kinase cascade, downstream of a variety of key regulators that respond to cell contact inhibition, transmembrane receptors, and unknown factors. The most critical effectors of the Hippo pathway are the Yes-activated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ). These transcriptional activators regulate the expression of multiple Hippo pathway targets.4 To successfully localize to the nucleus, YAP/TAZ must remain unphosphorylated; therefore, the Hippo pathway kinase cascade must be effectively “turned off” for YAP/TAZ nuclear translocation and activation of a predominantly proproliferative transcriptional program.4,7
YAP-1 expression is mainly controlled by the transcriptional activity of related target genes via the TEA domain/transcription enhancer factor family of transcription factors. Elevated YAP-1 expression has been observed in many human epithelial malignancies as such breast, liver, gastric, and esophageal cancers.4,8–10 However, there have been few reports concerning YAP-1 elevation in PTC, which is an epithelial cell-derived malignancy.
The present study evaluated YAP-1 expression in patient-derived PTC tumors, and assessed the associations of YAP-1 expression with clinicopathologic risk factors and prognosis [recurrence-free survival (RFS)] and assessed whether YAP-1 expression could be a prognostic marker.
MATERIALS AND METHODS
In total, 240 patients with histologically confirmed PTC, who were treated between December 2003 and January 2010 at the Department of Breast and Thyroid Surgery, Union Hospital Wu Han, China, were reviewed retrospectively. Complete clinicopathologic and prognostic data were available for all patients. Persistent disease/recurrence was assessed using imaging and was defined as the appearance of pathologically proven malignant tissue with or without metastatic lesions in the lungs, bones, or brain. Ethical approval for this study was granted by the Union Hospital Ethics Committee and informed consents of all patients were written.
Tissue Samples and Immunohistochemistry (IHC)
Paraffin-embedded tumor tissues were sliced into 3 to 4 μm continuous sections. For IHC, tissue sections were first deparaffinized in xylene and rehydrated in descending alcohol dilutions, followed by antigen retrieval by boiling in 0.01 mol/L citrate buffer (pH 6.0) for 20 minutes. After cooling at 20°C, sections were treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase and then blocked with goat serum [1:10 dilution in 0.1 M phosphate-buffered saline (PBS; pH 7.4)] for 20 minutes at 20°C. Sections were then incubated in primary YAP-1 antibody (#ab52771, clone: EP1674Y, dilution, 1:1000; CST, Boston, MA) overnight at 4°C. Whole tissue sections of YAP-1 nuclear-positive PTC tissue were used as positive tissue controls, whereas negative controls were performed by omitting the application of primary antibody replacing with PBS. The slides were washed 3 times in 0.1 M PBS (pH 7.4) for 3 minutes and then incubated with a biotinylated horseradish peroxidase goat anti-mouse/rabbit secondary antibody for 20 minutes at 20°C. Thereafter, the sections were incubated in 0.05% diaminobenzidine in 0.01 M PBS (pH 7.4) for 5 minutes at 20°C and counterstained with hematoxylin for visualization. Finally, sections were dehydrated in descending alcohol dilutions, mounted, and coverslipped. All slides were processed by the same pathologist.
The histopathologic examinations sections were stained with hematoxylin and eosin. Two independent pathologists reviewed all histologic sections and evaluated the IHC staining results according to the criteria of the World Health Organization.11,12 In cases of disagreement, the results were reanalyzed until a consensus was reached.
IHC staining (including fine immunoreaction areas existing in the negative external controls) was processed and evaluated by 3 experienced pathologists, blinded to the clinical data. Immunohistochemical staining for nuclear YAP-1 was evaluated using quick score (QS), a double-grading system.13 The QS represents the sum of a proportional score (PS), and intensity score. The PS was calculated as the ratio of nuclear YAP-1 immunopositive tumor cells to the total number of tumor cells. The PS was classified as follows: 0, 0% to 5%; 1, 6% to 25%; 2, 26% to 50%; 3, 51% to 75%; and 4, 76% to 100%. The intensity score was classified as follows: 0, no immunostaining at high magnification; 1, immunostaining only visible at high magnification; 2, immunostaining readily visible at low magnification; and 3, immunostaining strongly visible at low magnification. The QS score as 0 scores were defined as negative expression, 1 to 3 scores were defined as weak expression, 4 to 6 scores were defined as moderate expression, and 7 scores were defined as positive expression. Furthermore, we defined 0 to 3 scores as negative YAP-1 expression and 4 to 7 scores as positive YAP-1 expression for statistical analysis (Fig. 1). The slides were captured using a ScanScope slide scanner (Aperio, San Diego, CA), and representative images were obtained using Image Scope (Aperio) and Illustrator (Adobe, San Jose, CA) software.
EpiData software (version 3.1; EpiData Association, Odense, Denmark) was used for entry of initial clinical and pathologic data. Statistical analyses were performed using SPSS software (version 13.0; IBM SPSS Inc., Chicago, IL). Discrete variables were reported a proportion and analyzed by the χ2 test or Fisher exact test where appropriate. Kaplan-Meier survival curves and log-rank statistics were employed to evaluate RFS. Univariate and multivariate regression analyses were performed using the Cox proportional hazards model. All tests were 2-sided and P-values <0.05 were considered statistically significant.
Clinicopathologic Characteristics of the PTC Cases
Examples of positive and negative YAP-1 staining are shown in Figure 1. The clinicopathologic characteristics of the PTCs are listed in Table 1. The mean follow-up duration was 55.37±5.78 months (range, 18 to 95 mo). YAP-1 expression was positive in 149/240 tumors (62.1%) and negative in 91/240 tumors (37.9%). Fifty patients (20.8%) were male, and 190 patients were female (79.2%). Patients aged below 45 years and 45 years and above accounted for 32.1% and 67.9%, of the cohort, respectively. Lymph node metastasis was observed in 30.4% of patients, and nonlymph node metastasis was observed in 69.6%. Extrathyroidal extension was noted in 25% of patients and nonextrathyroid extension was noted in 75% of patients.
Associations Between YAP-1 Expression and Clinical Aggressiveness of PTC
There was a significant positive correlation between YAP-1 expression and larger tumor size (P=0.003), lymph node metastases (P=0.001), extrathyroidal extension (P=0.017), and tissue infiltration (P=0.012). None of the other clinicopathologic characteristics were associated with YAP-1 expression (Table 1).
Correlations of YAP-1 Expression With RFS
In patients with PTC, YAP-1 expression was significantly associated with inferior RFS (P=0.013; Fig. 2). In subgroup analyses, YAP-1 expression was significantly associated with RFS in females (P=0.008), in patients with lymph node metastasis (P=0.03), and in those with unifocal tumors (P=0.029; Figs. 3–5).
According to univariate analysis, YAP-1 expression, age, extrathyroidal extension, lymph node metastases, tissue infiltration, subtype, and larger tumor size were all associated with RFS in patients with PTC (Table 2). Multivariate analysis revealed that the independent predictors of RFS in patients with PTC included YAP-1 expression (P=0.039) and extrathyroidal extension (P=0.012).
Following curative resection, the overall survival rate for patients with PTC is good14; however, new biomarkers to identify patients at risk of recurrence would help to select patients who would benefit from more regular follow-up.
YAP activation has been associated with both epithelial cell tumors (breast, liver, and gastrointestinal tract) and stromal tumors (soft tissue sarcoma). Boin et al15 demonstrated that tumor cell proliferation in human schwannomas was linked with a signaling network controlled by YAP. Bai et al16 indicated that YAP functioned as a tumor suppressor that enhanced apoptosis by p53 modulating during chemotherapy. In nude mice xenografted with breast cell lines, YAP knockout in the cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxols, and enhanced tumor growth.17 However, the various conclusions drawn from previous reports suggest that there is no consistent role for YAP across different tumor types. Therefore, we evaluated if YAP expression represented a potential prognostic biomarker in PTC.
In the present study, almost two thirds of PTC tumors expressed YAP-1. Furthermore, YAP-1 expression was significantly associated with larger tumor size. This would be consistent with preclinical observations that YAP overexpression increased organ size in Drosophila and cell density saturation in murine NIH-3T3 fibroblast cell cultures.18 Furthermore, transgenic mice with liver-specific YAP overexpression showed a dramatic increase in liver size and eventually developed liver tumors.19,20 The mechanism underlying increased proliferation could be that YAP interacts with TEA domain/transcription enhancer factor transcription factors to induce epithelial-mesenchymal transition, which is often associated with cancer metastasis.21 Xia et al22 have illustrated that YAP promotes ovarian cancer cells proliferation and tumorigenesis in vitro and in vivo, and YAP was directly related to ovarian cancer development or progression. In the present study, YAP expression was also significantly associated with aggressive features such as extrathyroidal extension, lymph node metastasis, and tumor infiltration, indicating that YAP-expressing tumors represent more aggressive, advanced-stage tumors.
Clinically, RFS are the best-established predictive factors for important aspects response the prognosis of patients with PTCs. Previous studies have suggested that YAP-1 expression is associated with poor prognosis in various solid tumors including ovarian cancer,23 colorectal cancer,24 and urinary bladder cancer.25 In the present study, YAP-1 expression was associated with significantly inferior RFS in patients with PTC. In addition, YAP-1 and extrathyroidal extension were significant independent predictors of RFS. These findings would support the function of YAP-1 as an oncogene and a putative prognostic biomarker of recurrence in patients with PTC.
Serrano et al26 reported that the tyrosine kinase inhibitor dasatinib prevented the activity of YAP/TAZ by activation of core kinases such as MST1 and LATS, which subsequently inhibited the development of malignant tumors. Therefore, it is possible that inhibition of YAP-1 expression or action could potentially provide a novel drug target for molecular-targeted therapy in PTC.
This study included several important limitations. First, it used a retrospective single-center design. Second, we only evaluated associations with RFS because overall survival data were not available. Therefore, a prospective multicenter study with long-term follow-up is needed to confirm whether YAP-1 is an independent prognostic biomarker in patients with PTC.
YAP-1 expression was significantly correlated with high-risk clinicopathologic features and inferior RFS in patients with PTC. YAP-1 might represent a potential biomarker of PTC progression and a putative target for the development of novel anticancer treatments for PTC.
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Keywords:Copyright 2019 Wolters Kluwer Health, Inc. All rights reserved.
Yes-activated protein; immunohistochemistry; prognosis; papillary thyroid carcinoma