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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 8  |  Page : 114-119

Concomitant high expression of survivin and vascular endothelial growth factor-C is strongly associated with metastatic status of lymph nodes in papillary thyroid carcinoma


1 Department of Endocrinology and Radioimmunology, Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Belgrade, Serbia
2 Center for Endocrine Surgery, Institute for Endocrinology, Diabetes and Diseases of Metabolism, Clinical Center of Serbia, Belgrade, Serbia
3 Institute of Pathology, Medical Faculty, University of Belgrade, Belgrade, Serbia

Date of Web Publication26-Mar-2018

Correspondence Address:
Dubravka Cvejic
Institute for the Application of Nuclear Energy - INEP, 11080 Zemun, Belgrade, Banatska 31b
Serbia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.163675

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 > Abstract 


Purpose: Papillary thyroid carcinoma (PTC) has a strong propensity to metastasize to regional lymph nodes which increases the risk of local-regional relapse and affects the course of the disease. Molecular pathogenesis of lymph node metastasis (LNM) is not yet fully understood. Survivin, a multifunctionale molecule involved in apoptosis, proliferation and angiogenesis, and vascular endothelial growth factor-C (VEGF-C) are suggested to be implicated in lymphatic metastases of human malignancies.
Materials and Methods: Expression of survivin and VEGF-C was examined by immunohistochemistry and Western blot in 75 cases of PTCs in relation to their LNM status. Additionally, survivin and VEGF-C were immunohistochemically analyzed in 15 primary PTCs paired with their metastatic tissue in lymph nodes.
Results: High expression of survivin and VEGF-C was found in 62.7% and 64.0% cases, respectively, with a positive correlation to each other (Spearman's correlation co-efficient = 0.878, P < 0.001). Expression levels of both proteins were significantly higher in patients with LNM than in those without LNM (P < 0.001). The rate of concomitant high expression of survivin and VEGF-C in patients with LNM involvement was 88.9% (P < 0.01). Metastatic tissue in lymph nodes expressed survivin and VEGF-C at the same high extent as their primary tumors.
Conclusion: Concomitant high expression of survivin and VEGF-C is closely associated with LNM status of PTC patients, which suggests their cooperation in the metastatic process. Evaluation of survivin and VEGF-C expression could be clinically significant in predicting the metastatic potential of PTC and subsequent treatment and follow-up of these patients.

Keywords: Lymph node metastasis, papillary thyroid carcinoma, survivin, vascular endothelial growth factor-C


How to cite this article:
Selemetjev S, Savin S, Paunovic I, Tatic S, Cvejic D. Concomitant high expression of survivin and vascular endothelial growth factor-C is strongly associated with metastatic status of lymph nodes in papillary thyroid carcinoma. J Can Res Ther 2018;14, Suppl S1:114-9

How to cite this URL:
Selemetjev S, Savin S, Paunovic I, Tatic S, Cvejic D. Concomitant high expression of survivin and vascular endothelial growth factor-C is strongly associated with metastatic status of lymph nodes in papillary thyroid carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 20];14:114-9. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/114/163675




 > Introduction Top


Malignant tumors of the thyroid gland originating from follicular epithelium are generally classified as differentiated (papillary and follicular) and undifferentiated (anaplastic) carcinomas. The biological behavior of these subtypes of thyroid cancer is highly divergent, as reflected by differences in their patterns of metastases, clinical aggressiveness, and prognosis.[1],[2] Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignancy accounting for approximatey 80% of all thyroid carcinomas. PTC generally has a favorable outcome, but some patients develop local recurrence and/or distant metastases and die of their disease. On contrary to follicular thyroid carcinoma, which commonly spreads hematogenously, PTC has a strong propensity to metastasize to regional lymph nodes which increases the risk of local-regional relapse and affects the course of the disease.[3],[4],[5]

The clinical management of lymph node metastasis (LNM), including the extent of initial surgery and the proper indication for radioiodine therapy, is still controversial. This suggests that the prediction of LNM is important and requires better understanding of the underlying mechanism.

Molecular pathogenesis of LNM is not yet fully understood. Evidence shows that lymphangiogenesis is the key process involved in which new lymphatic vessels sprout from preexisting ones to facilitate the shedding of tumor cells into surrounding lymphatic vessels.[6] Recent evidence indicated that tumor lymphangiogenesis, that is, the growth of tumor-associated lymphatic vessels, promotes lymphatic metastasis.[7],[8]

There are several reports suggesting that survivin and vascular endothelial growth factor-C (VEGF-C) may take part in the course of lymphatic metastasis in human malignancies.

Survivin, a member of the apoptosis inhibiting proteins (IAPs) family [9] is a multifunctionale protein, which inhibits apoptosis, promotes proliferation, and facilitates angiogenesis.[10],[11] This protein could not be detected in normal adult tissues, but it has been found to be overexpressed in spontaneous human tumors and associated with aggressive tumor behavior and a worse outcome of patients.[12] Especially, it has been shown that survivin expression is significantly related to lymphatic metastasis in human carcinomas and that overexpression of this protein and its relation to the occurrence of LNM is a prognostic marker for these tumors.[13],[14],[15],[16],[17]

VEGF-C, a member of the VEGF family of angiogenic proteins, is recognized as a major lymphangiogenic factor. It has been found to induce lymphangiogenesis via activation of its receptor (VEGF receptor-3) expressed on endothelial cells of lymphatic vessels.[18] VEGF-C mediated lymphangiogenesis has been demonstrated to promote lymphatic metastasis.[8] With regards to thyroid tumors, the overexpression of VEGF-C has been observed in papillary thyroid cancer and associated with a high prevalence of LNM.[19],[20],[21],[22],[23]

Since it has been suggested that both, survivin and VEGF-C, play important roles in tumor lymphatic metastasis, in this study we evaluated, for the first time, expression of both molecules in relation to metastatic status of PTC.


 > Materials and Methods Top


Tissue samples

Archival (formalin-fixed, paraffin-embedded) tissues and fresh surgically removed tissues, from patients who had undergone surgery for PTC, were obtained from the Center for Endocrine Surgery, Institute of Endocrinology, Diabetes and Diseases of Metabolism, Clinical Center of Serbia, Belgrade, Serbia.

Histological slides from the thyroid tumor tissues stained by hematoxylin and eosin were reevaluated by the pathologist to confirm the diagnosis based on the World Health Organization standards.[24] Only common PTC cases were selected while rare histological subtypes (columnar, tall cell, insular, etc.) were excluded.

Clinical information obtained by reviewing the pathology reports included age, sex, tumor size, presence of LNM, and extrathyroid invasion (EI).

The study included a total of 75 patients diagnosed as having PTC. Among them 29 patients were younger and 46 older than 45 years (range: 11–76 years, mean: 48 years), and there were 59 females and 16 males. Tumor size ranged from 0.3 to 10 cm (mean: 28 mm). LNM were present in 27 cases at the time of surgery while EI was detected in 28 cases. The cases were thereafter classified into groups according to primary tumor status (pT) and primary tumor node metastasis (pTNM) stage based on the pTNM classification proposed by American Joint Committee on Cancer/Union for International Cancer Control.[25] There were 45 (67.31%) patients staged as I-II and 30 (32.69%) patients staged as III-IV.

The study protocol was approved by the Ethics Committee at the Center for Endocrine Surgery, Clinical Center of Serbia, Belgrade, Serbia.

Immunohistochemistry

Monoclonal antibody against survivin (D-8, sc-17779, Santa Cruz Biotechnology, Inc., California, USA) and rabbit polyclonal antibody against VEGF-C (Abcam plc, Cambridge, UK) were used for immunohistochemistry.

A positive reaction was identified using a streptavidin-biotin-peroxidase detection system supplied by Vector Laboratories (Burlingame, CA, USA).

Tissue sections (4–6 μm thick) from each block were deparaffinized with xylene and rehydrated through a series of descending graded ethanol. Endogenous peroxidase activity was blocked with 0.3% H2O2/methanol for 30 min followed by incubation with nonimmune horse serum for 20 min to block nonspecific binding. Tissue sections were then incubated with primary antibody against survivin (1: 300 dilution) or VEGF-C (1:50 dilution) at 4°C overnight. This was followed by incubation with biotinylated horse anti-mouse IgG (for survivin) or with biotinylated goat-anti rabbit IgG (for VEGF-C) for 30 min and thereafter with the avidin-biotin-peroxidase complex for 30 min. Between each step, sections were washed 3 times in phosphate buffered saline (PBS). The reaction was visualized using 3, 3'-diaminobenzidine tetra hydrochloride solution as the chromogen.

After counterstaining with hematoxylin, slides were dehydrated in ascending ethanol, cleared with xylene, mounted with coverslips using a permanent mounting medium and thereafter examined using an Axio Imager 1.0 microscope (Carl Zeiss, Jena, Germany) with a Canon A640 Digital Camera System.

Controls were incubated with PBS in place of the primary antibody and no positive staining was observed.

Scoring of immunohistochemical staining

Cytoplasmic staining for survivin or VEGF-C was scored by two independent observers as follows: (0) Absence of staining, (1) weak widespread or focal (up to 40%) staining of tumor cells, (2) moderate staining in more than 40% of tumor cells and (3) strong staining in more than 40% of tumor cells. Cases scored as (0) and (1) were combined in a low expressing group, and those scored as (2) and (3) in a high expressing group.

Statistical data

Statistical analysis was performed with SPSS software (version 11.5, SPSS Inc., Chicago, IL, USA) using Fisher's exact test and Spearman's correlation analysis. P < 0.05 were considered as statistically significant.

Protein extraction

Proteins were extracted from snap-frozen normal and malignant thyroid tissues by initially cutting 100 mg portions into small pieces and resuspending in 1 ml of extraction buffer (20 mM Tris HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM ethylenediaminetetraacetic acid) containing a complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA). The specimens were then homogenized for 30 min on ice and centrifuged for 20 min at 14,000 rpm at 4°C. The supernatant was aliquoted and the protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA).

Western blotting

Protein samples (100 μg) from supernatants of the tumor and nonmalignant homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 15% gels. After electrophoresis, proteins were transferred onto PVDF membrane according to the manufacturer's instructions (Millipore, Billerica, MA, USA). The membrane was blocked for 2 h with 5% casein in TBS-T at room temperature and then incubated with primary antibody in the blocking solution (1:5000 dilution) overnight at 4°C. The primary antibodies to survivin or VEGF-C were the same as those used for immunohistochemistry. After extensive washing with TBS-T, the membrane was probed with biotinylated horse anti-mouse IgG (for survivin) or goat-anti-rabbit IgG (for VEGF-C) at 1:2500 dilution for 35 min at room temperature. After further washes with TBS-T, the next incubation was in ABC solution for 30 min at room temperature, followed by visualizing immunoreactive products with enhanced chemiluminescence Substrate (Pierce, Rockford, IL, USA).


 > Results Top


Immunohistochemical results

Survivin and VEGF-C protein expression were evaluated by immunohistochemistry in 75 formalin-fixed, paraffin-embedded tissue sections from patients diagnosed with PTC. Immunohistochemical staining results are shown in [Table 1]. Survivin and VEGF-C expressions were localized in the cytoplasm of malignant cells. When present, nonmalignant (normal or hyperplastic) tissue adjacent to papillary carcinoma was negative [Figure 1]. Both proteins were expressed in 68 out of 75 cases analyzed at score 1–3 for 28.1%, 25.3%, and 37.3% out of the total number of positive cases for survivin and 26.7%, 34.7%, and 29.3% respectively for VEGF-C. There were 47 (62.7%) high expressing cases of PTC for survivin (cases scored as 2 and 3) and 48 (64.0%) high expressing cases for VEGF-C. We observed a correlation between expression levels of survivin and VEGF-C, which appeared in most cases as almost identical immunostaining pattern [Figure 1]. Statistical analysis of the relation between survivin and VEGF-C expressions confirmed significance by Spearman's correlation test (r = 0.878, P < 0.001).
Table 1: Immunohistochemical expression of survivin and VEGF-C in papillary thyroid carcinoma (n=75)

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Figure 1: Strong immunohistochemical staining for survivin (a and c) and vascular endothelial growth factor-C (b and d) in two cases of papillary thyroid carcinoma with lymph node metastasis involvement

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Correlation between survivin and vascular endothelial growth factor-C expression and lymph node metastasis

[Table 2] shows the association between immunohistochemical expression of survivin and VEGF-C and lymph node status of PTCs. High expression of survivin correlated significantly with the presence of LNM (P = 0.0004,) with a positive rate of 88.9%. Similarly, high expression of VEGF-C was closely correlated with metastatic PTC (P = 0.0001) at a positive rate of 92.6%. Coexpression of high levels of both survivin and VEGF-C was found in 24 out of 27 metastatic PTCs giving a positive rate of 88.9% (P = 0.0002).
Table 2: Survivin and VEGF-C immunohistochemical expression in relation to LNM status of PTC (n=75)

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Thus, high expression of a single protein (survivin or VEGF-C) or coexpression of high levels of both (due to their strong correlation to each other) was significantly associated with positive lymph node metastatic status in PTC patients.

Western blot analysis of survivin and vascular endothelial growth factor-C expression in papillary thyroid carcinoma

Survivin and VEGF-C expression was analyzed in malignant papillary carcinoma tissue and corresponding normal tissue by Western blot, to confirm the specificity of primary antibodies and to extend immunohistochemical data. Representative immunoblots from paired tumorous and nontumorous tissues obtained from PTC patients are shown in [Figure 2]. Survivin expression was up-regulated in malignant tissue, while it was not detected in its nonmalignant counterpart. Furthermore, stronger survivin expression was observed in PTCs with LNM than in those without LNM. VEGF-C expression level was also notably higher in the tumor group, particularly in the PTC with LNM, than in nonmetastatic carcinomas.
Figure 2: Western blot analysis of survivin and vascular endothelial growth factor-C in representative panels of papillary thyroid carcinoma tissues (T) and matched nonmalignant tissues (N). Higher expression of survivin and vascular endothelial growth factor-C in papillary thyroid carcinomas with lymph node metastasis than in papillary thyroid carcinomas without lymph node metastasis

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Correlation between survivin and vascular endothelial growth factor-C expression in tumor tissues and their lymph node metastases

To determine whether the expression of survivin or VEGF-C differs between pTs and their LNM, we immunohistochemically analyzed 15 papillary carcinoma tissues paired with their metastatic tissue in lymph nodes.

As shown in [Figure 3], all pTs expressed high levels of survivin and VEGF-C (scores 2 or 3). In the lymph node tissues, both survivin and VEGF-C were detected at similar levels as in their pTs [Figure 4].
Figure 3: Graphical illustration of survivin and vascular endothelial growth factor-C expression in 15 cases of papillary thyroid carcinomas and their matched lymph node metastases. Vertical axis: Immunohistochemical scores for survivin or vascular endothelial growth factor-C (0–3; as detailed in the Material and Methods section)

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Figure 4: Immunohistochemical expression of survivin and vascular endothelial growth factor-C in two cases of primary papillary thyroid carcinoma and corresponding metastatic tissue in lymph nodes. Immunostaining for survivin (a) and vascular endothelial growth factor-C (c) in the primary tumors and paired lymph node metastases (b and d)

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 > Discussion Top


Thyroid carcinomas metastasize by different mechanisms comprising direct invasion of the surrounding tissue and spreading via the lymphatic or vascular system. Among differentiated carcinomas of the thyroid gland, PTC has a strong propensity to metastasize to regional lymph nodes while follicular thyroid carcinoma metastasizes by a hematogenous rather than a lymphatic course.

Despite their clinical relevance, the molecular mechanisms that guide the route of spreading and localization of the metastases in different tissues are not completely understood.

A lot of evidence documented that lymphangiogenesis is the key process involved in lymph node metastatic dissemination. VEGF-C is one of the most potent factors known to induce tumor lymphangiogenesis and promote LNM.[7],[8]

Clinically, up-regulation of VEGF-C has been observed in different tumor types, and its overexpression has been associated with a high prevalence of LNM.[26] With regards to the thyroid, several studies have shown a significant relationship between VEGF-C expression and regional LNM. The presence of metastatic lymph nodes was found to be positively correlated with expression of VEGF-C in primary thyroid carcinomas prone to lymph node invasion.[19],[20],[21],[22] Fellmer et al. reported a higher level of VEGF-C mRNA in PTC, but not in follicular thyroid carcinoma, as shown by in situ hybridization or quantitative PCR assay.[20] de la Torre et al.[23] observed an increased lymph vessel density and up-regulation of VEGF-C expression in PTC when compared to benign lesions or follicular thyroid carcinoma. These results emphasized VEGF-C induced lymphangiogenesis as an important mechanism underlying the metastatic behavior of PTC.

Additionally, several studies have shown that a number of molecular markers in the pT may be predictive for lymph node metastatic dissemination.

Survivin, which was firstly identified as a member of the IAP family of proteins,[9] appeared to act multifunctionally, not only inhibiting apoptosis but also promoting proliferation, angiogenesis, and metastatic dissemination.[9],[10],[11] High levels of survivin have been detected in various human tumors and associated with LNM and aggressive tumor behavior.[12] With regards to the thyroid, it has been reported that up-regulation of survivin expression is an early event in thyroid tumorigenesis [27] with further increase in correlation with the invasion, metastasis and tumor progression.[28],[29],[30]

In this study we analyzed, for the first time, expression of both proteins, survivin, and VEGF-C, in a series of PTC patients in relation to their LNM status. Our results show that expression levels of both proteins were significantly higher in patients with lymphatic invasion than in patients without lymphatic invasion. Furthermore, concomitant high expression of survivin and VEGF-C was found in metastatic tissue in lymph nodes. These results indicate an obvious link between survivin and VEGF-C and suggest their cooperation in the metastatic process.

Coexpression of survivin and VEGF-C and their association with LNM was recently shown in breast carcinoma tissue by Cai et al.[31] and in gastric carcinoma tissue by Zhang et al.[32] By manipulating production of survivin and VEGF-C in cell lines, these authors further demonstrated that survivin expression induces up-regulation of VEGF-C, indicating that survivin is a regulator of VEGF-C expression in breast and gastric carcinoma cells and is essential for invasion and lymphatic metastasis in these cancers.

Our finding here of a significant correlation between survivin and VEGF-C levels is consistent with the coordinated regulation of expression of these two proteins proposed for breast and gastric cancer.

Further evidence supporting a role for survivin in invasion and metastasis has been provided from a mouse model of breast carcinoma in which a survivin dominant mutant (mutation of the threonine residue at position 34) inhibited tumor growth and LNM.[33] However, molecular mechanisms underlying survivin role in invasiveness and metastasis are not yet fully understood. Mehrotra et al.[34] recently showed that molecular interaction between survivin and XIAP (another member of the IAP family) stimulates tumor cell invasion and promotes metastasis through a pathway independent of IAP inhibition of cell death.

The relationship between survivin expression and metastasis may also arise from an essential function of survivin, that is inhibition of apoptosis. The anti-apoptotic effect of survivin is not confined only to cancer cells, but can also be manifested in endothelial cells. In that way, VEGF-C and survivin appear to function cooperatively to increase and maintain newly formed lymphatic vessels. Thus, molecular mechanisms that guide the route of spreading and localization of a metastasis are rather complex, but better understanding is relevant for both tumor prognosis and treatment.


 > Conclusion Top


This study reports a strong association between concomitant high expression of survivin and VEGF-C and the metastatic status of lymph nodes in PTC patients, which suggests the possibility of their cooperation in the metastatic process. Evaluation of survivin and VEGF-C expression could be clinically significant in predicting the metastatic potential of PTC and subsequent decisions about the treatment and follow-up of these patients.

Financial support and sponsorship

The Ministry of Education, Science and Technological Development of the Republic of Serbia, Project 173050: “Molecular characterization of thyroid gland tumors: Biological and clinical aspects”.

Conflicts of interest

There are no conflicts of interest.



 
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