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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3694-3699
Copyright © 2003 by The Endocrine Society

Expression of Vascular Endothelial Growth Factor-C in Benign and Malignant Thyroid Tumors

Chung J. Hung, David G. Ginzinger, Raza Zarnegar, Hajime Kanauchi, Mariwil G. Wong, Electron Kebebew, Orlo H. Clark and Quan-Yang Duh

Endocrine Surgical Oncology Fellows (C.J.H., R.Z., H.K.), Department of Surgery (M.G.W., E.K., O.H.C., Q.Y.D.), University of California San Francisco (UCSF)/Mount Zion Medical Center, Genome Analysis Core Facility (D.G.G.), Cancer Center, UCSF, San Francisco, California 94143; Surgical Service (Q.Y.D.), Veterans Affairs Medical Center, San Francisco, California 94121; and Department of Surgery (C.J.H.), National Cheng Kung University Hospital, Tainan 70441, Taiwan

Address all correspondence and requests for reprints to: Quan-Yang Duh, M.D., Surgical Service, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: duhq{at}surgery.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In contrast to vascular endothelial growth factor (VEGF), which stimulates angiogenesis, VEGF-C is thought to stimulate lymphangiogenesis. The role of VEGF-C in thyroid cancer pathogenesis has not been clarified. One might expect a different pattern of VEGF-C expression in the various types of thyroid cancer because of their different means of metastases. In this investigation, we determined whether the differential expression of VEGF-C might explain the different propensity to lymph node metastasis in thyroid cancers. One hundred eleven normal and neoplastic thyroid tissues were analyzed by real-time quantitative PCR. Papillary thyroid cancers had a higher VEGF-C expression than other thyroid malignancies (P < 0.0005 ANOVA). Among the normal thyroid tissues from patients with malignant or benign thyroid diseases, there was no significant difference in VEGF-C expression. Paired comparison of VEGF-C expression between thyroid cancers and normal thyroid tissues from the same patients showed a significant increase of VEGF-C expression in papillary thyroid cancer (1.10 ± 0.41 vs. 0.70 ± 0.13; P = 0.001) and a significant decrease of VEGF-C expression in medullary thyroid cancer (0.11 ± 0.13 vs. 0.78 ± 0.29; P = 0.001). In contrast, there was no significant difference of VEGF-C expression between cancer and normal tissues in other types of thyroid cancer. In summary, VEGF-C expression is increased in papillary thyroid cancer, compared with paired normal thyroid tissues, but not in other thyroid cancers that are also prone to lymph node metastasis. The lymphangiogenic role of VEGF-C in thyroid cancers therefore appears to be complex and other factors are likely to be also involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LYMPH NODE METASTASIS is common in most cancers but the molecular mechanisms underlying lymph node metastasis are poorly understood. In contrast to vascular endothelial growth factor (VEGF), which stimulates angiogenesis, VEGF-C is thought to stimulate lymphangiogenesis. VEGF-C was found in the supernatant of prostate cancer cells in 1996 (1). VEGF-C is expressed in multiple human tissues, predominantly in the heart, placenta, muscle, ovary, and small intestine. VEGF-C binds to the VEGF receptor-3 (Flt-4), and the expression of VEGF receptor-3 is highly restricted to the lymphatic endothelium in adult tissues (2). Transgenic overexpression of VEGF-C in keratocytes caused lymphatic, but not vascular, endothelial proliferation and vessel enlargement in the mouse skin (3). Recombinant VEGF-C protein induced significant lymphangiogenesis, but only mild angiogenesis, in avian chorioallantoic membranes (4). Several studies have shown that cancers that metastasize to lymph nodes usually express more VEGF-C (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17), but this correlation is not universal (18, 19, 20, 21, 22). VEGF-C expression is up-regulated in many (5, 7, 9, 13, 22, 23, 24) but not in all human cancers (18, 19).

The role of VEGF-C in thyroid cancer pathogenesis has not been completely clarified. Different thyroid cancers have a different propensity for lymph node metastasis. For example, papillary thyroid cancer tends to metastasize to regional lymph nodes, whereas follicular thyroid cancer usually metastasizes by a hematogenous rather than by a lymphatic route. Other less common thyroid cancers, such as Hürthle cell cancers, medullary cancer, and anaplastic cancer also frequently metastasize to lymph nodes. In this investigation, we determined whether VEGF-C expression differed in various thyroid cancers that metastasize via a lymphatic or hematogenous route. We hypothesized that thyroid cancers that metastasize to lymph nodes express more VEGF-C than those that do not.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

One hundred eleven thyroid and lymph node tissues were obtained from patients who underwent thyroid operations at the University of California Mount Zion Medical Center, San Francisco (UCSF). The protocol was approved by the human research committee of UCSF. Tissues were obtained in the operating room after removal and immediately frozen in liquid nitrogen, and kept at -80 C until analysis. Tumors were histologically classified according to the World Health Organization (WHO) recommendations. The thyroid tissues included 15 papillary cancers, eight follicular cancers, six Hürthle cell cancers, 11 medullary cancers, three anaplastic cancers, six follicular adenomas, five Hürthle cell adenomas, four Graves’ disease, and 48 corresponding paired normal thyroid tissues in most of these patients when available (15 papillary cancers, six follicular cancers, six Hürthle cell cancers, seven medullary cancers, three anaplastic cancers, six follicular adenomas, five Hürthle cell adenomas). Tissues from metastatic lymph nodes were also obtained from five patients with papillary cancer.

RNA extraction and cDNA synthesis

Total RNA was extracted from tissue specimens using the TRIzol method (Life Technologies, Inc., Gaithersburg, MD) according to the manufacture’s suggested protocol. Concentration and purity was determined using a Beckman spectrophotometer. The quality of RNA was checked by the electrophoresis of 3-µg samples in a 1.5% agarose gel, staining with ethidium bromide. The 28S and 18S rRNA bands were examined on a UV transilluminator. No significant degradation was observed in any RNA samples.

Total RNA (250 ng) was reverse transcribed in a total reaction volume of 100 µl containing 250 U Maloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 40 U ribonuclease inhibitor (Roche, Nutley, NJ), 1 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) (Amersham Pharmacia Biotech Inc., Arlington Heights, IL), 7.5 mM MgCl2, 5 mM random hexamer (Invitrogen, Carlsbad, CA), and 1 x PCR buffer II (Applied Biosystems, Foster City, CA). Complementary DNA was synthesized using a thermal cycler (Amplitron II Thermolyne, Barnstead/Thermolyne, Conroe, TX) by incubating at 25 C for 10 min, 48 C for 40 min, and 95 C for 5 min.

Real-time quantitative PCR

Oligonucleotide primers and Taqman probes were designed to be intron spanning using Primer Express software (Applied Biosystems) and purchased from Biosearch Technology (Novato, CA). VEGF-C-specific primers, spanning exons 4 and 5, were designed to amplify a 93-bp product. A blast alignment of the VEGF-C Taqman amplicon indicates no significant sequence similarity to other related genes including VEGF-A-165. Human ß-glucuronidase (GUS) gene was used as an endogenous control to normalize the expression of VEGF-C (25). The sequences of the primers and probes of VEGF-C and GUS genes are shown at Table 1Go. Optimal PCR conditions and PCR efficiency were determined empirically and were more than 90% efficient.


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  		TABLE 1. Primer and probe sequences used in the real-time quantitative RT-PCR

 
Quantitative real time PCR was then performed using a 96-well optic tray on the ABI Prism 7700 sequence detection system (Applied Biosystems). A total reaction volume of 50 µl contained 1 x TaqMan buffer A (Applied Biosystems), 5.5 mM MgCl2, 200 µM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 1.25 U AmpliTaq Gold (Applied Biosystems), 500 nM of each primer, 200 nM probe, and 10 µl cDNA (described above, equivalent to a cDNA amount from 25 ng of initial total RNA). The PCR thermal cycle condition was set up at 95 C for 12 min followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min.

The mRNA content of each target gene was simultaneously determined in the 96-well. Each sample was run as a triplicate. The negative controls lacking template RNA were included in each experiment. Data collection and analysis was performed with SDS version 1.7 software (Applied Biosystems). Data were then exported and further analyzed in Excel (Microsoft, Redmond, WA).

The comparative cycle threshold (Ct) method

The comparative Ct method was used as previously described (26). Briefly, the difference in Ct, the number of PCR cycles required for the FAM (i.e. 6-carboxyfluorescein) emission intensities to exceed a threshold, between the VEGF-C and GUS genes was calculated and designated as {Delta}Ct. VEGF-C mRNA expression, relative to GUS, is calculated according to the following formula: 2-{Delta}Ct. These calculations are valid because the measured PCR efficiencies are very close to 100%.

Statistical analysis

All data are expressed as mean ± SEM. Differences among multiple groups were analyzed using ANOVA. The Tukey honestly significant difference test was used as the post hoc test. Differences between two groups were compared using unpaired t test. Paired analysis was performed by paired t test (SPSS, version 11.0; SPSS Inc., Chicago, IL). A P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VEGF-C mRNA expression

Papillary thyroid cancers had a higher VEGF-C expression than other thyroid malignancies (P < 0.0005, ANOVA) (Table 2Go). However, there was no significant difference between all malignant thyroid tissues and all benign thyroid tissues (P = 0.767). When compared with unpaired normal thyroid tissues (n = 48), there was an increase of VEGF-C expression in papillary thyroid cancers and a decrease of VEGF-C expression in Hürthle cell, medullary, and anaplastic thyroid cancers (Table 2Go). Among the normal thyroid tissues from patients with malignant or benign thyroid diseases, there was no significant difference in VEGF-C expression (Tables 3Go and 4Go). There was no difference in VEGF-C expression between patients younger than or older than 45 yr of age for the entire group of malignant neoplasms (0.70 ± 0.53 vs. 0.54 ± 0.49; P = 0.313) or the papillary thyroid cancers (1.13 ± 0.34 vs. 1.00 ± 0.61; P = 0.603).


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  		TABLE 2. VEGF-C expression, relative to GUS expression, in malignant and benign thyroid tissues

 

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  		TABLE 3. Paired comparison of VEGF-C expression, relative to GUS expression, between malignant thyroid tumors and normal thyroid tissues from the same patients

 

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  		TABLE 4. Paired comparison of VEGF-C expression, relative to GUS expression, between benign thyroid tumors and normal thyroid tissues from the same patients

 
VEGF-C expression in paired samples (Tables 3–6GoGoGoGo)

Samples from both malignant and normal thyroid tissue were available in 48 patients. In these paired-sample analyses, the normal thyroid tissue from the same patient served as a control because VEGF-C expression may be regulated by various factors, such as epidermal growth factor, that differ among the patients.


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  		TABLE 5. Paired comparison of VEGF-C expression, relative to GUS expression, in different subgroups of patients with PTC

 

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  		TABLE 6. Comparison of VEGF-C expression, relative to GUS expression, among PTC tissues, metastatic lymph nodes, and normal thyroid tissues from the same patients

 
Paired comparison of VEGF-C expression between thyroid cancers and normal thyroid tissues from the same patients showed an increase of VEGF-C expression in papillary thyroid cancers (1.10 ± 0.41 vs. 0.70 ± 0.13; P = 0.001) (Fig. 1AGo) and a decrease of VEGF-C expression in medullary thyroid cancers (0.11 ± 0.13 vs. 0.78 ± 0.29; P = 0.001) (Fig. 1BGo) (Table 3Go). In contrast, there was no difference of VEGF-C expression between cancer and normal tissues in other types of thyroid cancer (Fig. 1Go, C–E).



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  		FIG. 1. Paired comparison of VEGF-C expression, relative to GUS expression, between malignant thyroid tumors and normal thyroid tissues from the same patients (*, P < 0.05, paired t test). A, PTC, papillary thyroid cancer (n = 15); B, MTC, medullary thyroid cancer (n = 7); C, FTC, follicular thyroid cancer (n = 6); D, HTC, Hürthle cell thyroid cancer (n = 6); E, ATC, anaplastic thyroid cancer (n = 3).

 
Paired comparison of VEGF-C expression between benign thyroid tumors and normal thyroid tissues from the same patients showed a significant decrease of VEGF-C expression in Hürthle cell adenoma (0.52 ± 0.34 vs. 1.19 ± 0.33; P = 0.026) (Fig. 2BGo) but not in follicular adenoma (Fig. 2AGo) (Table 4Go).



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  		FIG. 2. Paired comparison of VEGF-C expression, relative to GUS expression, between benign thyroid tumors and normal thyroid tissues from the same patients (*, P < 0.05, paired t test). A, FA, follicular adenoma (n = 6); B,. HA, Hürthle cell adenoma (n = 5).

 
To determine whether different subgroups of patients with papillary thyroid cancer have different VEGF-C expression, we analyzed those with clinically positive cervical lymph node metastases, clinically negative cervical lymph node metastases, and follicular variants of papillary cancer (Table 5Go). There was a significant increase of VEGF-C expression in papillary thyroid cancer tissues from the node-positive group (1.25 ± 0.47 vs. 0.65 ± 0.15; P = 0.024) (Fig. 3AGo) and the node-negative groups (1.16 ± 0.44 vs. 0.67 ± 0.10; P = 0.027) (Fig. 3BGo). However, there was no significant difference in VEGF-C expression in a subgroup of patients with follicular variants of papillary thyroid cancer (0.81 ± 0.14 vs. 0.79 ± 0.12; P = 0.659) (Fig. 3CGo).



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  		FIG. 3. Paired comparison of VEGF-C expression, relative to GUS expression, in different subgroups of patients with papillary thyroid cancer (*, P < 0.05, paired t test). PTC, Papillary thyroid cancer; LN meta (+), positive cervical lymph node metastasis; LN meta (-), negative cervical lymph node metastasis; tumor, thyroid cancer tissue from the thyroid; normal, normal thyroid tissue from the same patient.

 
To determine whether VEGF-C expression differed between primary papillary thyroid cancers, metastatic lymph nodes, and normal thyroid tissues, we analyzed these three tissues from the five patients who presented with clinically positive cervical lymph node metastases (Table 6Go). VEGF-C expression in the primary thyroid cancers was higher, compared with either VEGF-C expression in the metastatic lymph nodes or normal thyroid tissues from the same patient (cancer 1.25 ± 0.47 vs. normal 0.65 ± 0.15 vs. lymph node 0.43 ± 0.20; P = 0.003) (Fig. 4Go).



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  		FIG. 4. Comparison of VEGF-C expression, relative to GUS expression, among papillary thyroid cancers tissues, metastatic lymph nodes, and normal thyroid tissues from the same patients (*, P < 0.05, ANOVA). Tumor, thyroid cancer tissue from the thyroid; normal, normal thyroid tissue; LN, thyroid cancer tissue from metastatic lymph node (n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous investigations have previously documented a significant correlation between VEGF-C expression in primary cancer tissues and the occurrence of lymph node metastases in thyroid cancers (5, 6), head and neck squamous cell carcinomas (7), lung (8), esophageal (9), gastric (10, 11, 12), pancreatic (13), colorectal (14), prostate (15), and cervical cancers (16, 17). However, this association of VEGF-C with lymphatic metastasis is not universally observed in all investigations. In some studies in lung cancers (18), breast cancers (19), mesothelioma (20), neuroblastomas (21), and colorectal cancers (22), there were no direct correlation between VEGF-C expression in primary cancers and lymph node metastases. The reason for the differences is not clear but may relate to sample preparation, different experimental techniques, or the differences in tumor histology.

Theoretically one would expect higher VEGF-C expression in papillary, Hürthle cell, medullary, and anaplastic thyroid cancers than in follicular thyroid cancers because these cancers metastasize to regional lymph nodes. In our investigation, we found higher VEGF-C expression in papillary thyroid cancers. Surprisingly, however, we found a lower expression of VEGF-C in Hürthle cell, anaplastic, and medullary thyroid cancers. What we found is in part similar to one previous report showing a significantly higher VEGF-C gene expression in papillary than in follicular thyroid cancers (27). It is, however, contrary to another study (5) that reported a generalized up-regulation of VEGF-C mRNA in papillary thyroid cancers (76%), undifferentiated thyroid cancers (86%), and medullary thyroid cancers (83%), when compared with unpaired normal thyroid tissues.

We also found no significant difference in VEGF-C expression in papillary thyroid cancers from patients who had or did not have cervical lymph node metastases, although both groups showed up-regulation of VEGF-C expression in the thyroid cancer tissues when compared with paired normal thyroid tissues from the same patients.

One previous study reported an up-regulation of VEGF-C mRNA levels in metastatic lymph nodes in patients with papillary thyroid cancers (92%) and medullary cancers (100%), compared with unpaired normal thyroid tissues (5). In contrast, when examining the VEGF-C expression of metastatic lymph nodes five papillary thyroid cancer patients with clinically positive cervical lymph node metastases, we found that the VEGF-C expression in either metastatic lymph nodes or paired normal thyroid tissues was significantly lower than that found in the paired primary thyroid cancers from the same patients.

There have been conflicting reports about the expression of VEGF-C in human primary (not lymph node) cancers. VEGF-C expression was found to be increased in head and neck squamous cell carcinomas (7, 23), esophageal squamous cell carcinomas (9), colorectal cancers (22), pancreatic cancers (13), thyroid cancers (5), and cervical cancers (24). The expression was found to decrease in lung adenocarcinoma (18) and showed no difference in breast cancer (19).

One of the reasons for the conflicting results may be because nonpaired normal tissue was used for the comparison in these studies. Paired-sample analysis, using the tumor and normal thyroid tissues from the same patients, controls for factors that regulate VEGF-C expression that may differ from patient to patient. VEGF-C mRNA expression is up-regulated by serum and various growth factors, such as platelet-derived growth factor, epidermal growth factor, TGFß and the tumor promoter phorbol myristate 12,13-acetate (28). IL-1ß and TNF{alpha} also up-regulate VEGF-C mRNA expression in human lung fibroblasts and umbilical vein endothelial cells (29). In contrast, in our investigation, TSH, which stimulates the expression of many genes in thyroid cells, did not stimulate VEGF-C mRNA expression in thyroid cancer cell lines (unpublished data).

Interestingly, although we found increased VEGF-C expression in papillary cancers and decreased VEGF-C expression in medullary cancers, we found no differences in cancers and normal tissues from Hürthle cell and anaplastic thyroid cancers (although a tendency to be lower in cancers). It is possible that the lower expression of VEGF-C in Hürthle cell and anaplastic thyroid cancers might become statistically significant if more samples are studied. Taken together our results demonstrate that lymphangiogenesis is a complex process and the expression of VEGF-C is not the only mechanism in lymphangiogenesis and lymph node metastasis. Other factors are likely to be involved.

As predicted, follicular cancers and adenomas did not have an increased VEGF-C expression. Interestingly, the few follicular variants of papillary thyroid cancer that we studied, also showed no increase in contrast to other types of papillary thyroid cancer.

Inhibiting VEGF activity using monoclonal antibodies can reduce the growth and angiogenesis in cancers (30). Lymphangiogenesis may similarly be a new target for anticancer therapy. Inhibiting VEGF-C activity in cancers that express high levels of VEGF-C may reduce lymph node metastasis. For example, one might speculate that papillary thyroid cancer, but not the Hürthle cell, anaplastic, or medullary thyroid cancer, would be a good candidate for such treatment.

In summary, VEGF-C expression is increased in papillary thyroid cancer, compared with paired normal thyroid tissues, but not in other thyroid cancers that are also prone to lymph node metastasis. Therefore, the lymphangiogenic role of VEGF-C in thyroid cancers is complex and other factors are likely to be also involved.


   Footnotes
 
This work was supported in part by the Jerold Heller Family Foundation, the Sanford Diller Family Foundation, Ministry of Education in Taiwan, Friends of Endocrine Surgery, and Mt. Zion Health Systems.

Abbreviations: Ct, Cycle threshold; GUS, ß-glucuronidase; VEGF-C, vascular endothelial growth factor-C.

Received January 16, 2003.

Accepted May 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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