Abstract
Vascular endothelial growth factor (VEGF) is a potent stimulator for angiogenesis. It has been implicated in growth and metastasis of thyroid cancer. Three functional single nucleotide polymorphisms (SNPs) of VEGF (−2578C/A, −634G/C, and +936C/T) are known to be related with VEGF expression. We conducted a case–control study to evaluate the genetic effects of these three functional SNPs on the development of thyroid cancer and lymph node metastasis. A total of 332 cases and 261 controls were recruited for this study. The genotypes were determined by the TaqMan 5′-nuclease assay. Hardy–Weinberg equilibrium (HWE) was tested for each SNP, and genetic effects were evaluated by the χ2-test and multiple logistic regression. We used Bonferroni correction to account for multiple testing, and a two-tailed P value <0.017 was considered statistically significant. All three SNPs were in HWE. The A allele of −2578C/A (i.e. SNP rs699947) increased a risk for thyroid cancer (adjusted OR=136, 95% CI=1.02–1.81, P=0.039). Haplotype analysis yielded a less significant result (an empirical P value of 0.07). There was a tendency of increasing the frequency of the risk allele from controls, patients without lymph node metastasis to patients with lymph node metastasis (Ptrend=0.019). Further analysis showed that the genetic effect was only in men (adjusted OR=1.97, 95% CI=1.16–3.37, P=0.013) but not in women (adjusted OR=1.15, 95% CI=0.81–1.62, P=0.435). The other two SNPs did not show significant results. The A allele of the SNP rs699947 increased the risk of thyroid cancer development and regional lymph node metastasis in men.
Introduction
Thyroid cancer accounts for <1% of all human cancers but is the most common endocrine neoplasia (Schlumberger & Torlantano 2000). The incidence of thyroid cancer (0.5–10/ 100 000) increases with age, and it reaches a plateau after the age of 50 years (DeLellis et al. 2004). Thyroid cancer is two to four times more frequent in females than males. Radiation exposure, hormonal factors, and family history are the risk factors for thyroid carcinoma (Kondo et al. 2006). Genetic predisposition has been proposed as one of the risk factors for thyroid cancer (Malchoff & Malchoff 2006). Different histological types of thyroid cancer have distinct clinical features. More than 90% thyroid tumors are well-differentiated carcinoma, including papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). For these two types of thyroid cancer, the treatment can be effective and prognosis is often favorable for patients identified in the early stage.
In contrast, anaplastic thyroid cancer is aggressive, responds poorly to treatment, and is associated with worse prognosis (Kondo et al. 2006). Follicular thyroid carcinoma tends to spread via the blood stream, whereas papillary thyroid tumor spreads occur via the lymphatic system (Turner et al. 2003).
Angiogenesis is a process of new blood vessel development from preexisting vasculature. The vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a crucial role in both angiogenesis and lymphogenesis (Carmeliet & Jain 2000). The microvascular density is increased in the thyroid malignancy compared with normal thyroid tissue and benign thyroid tumors (Segal et al. 1996, Akslen & Livolsi 2000). Growing evidence from in vitro and in vivo experiments have shown that increased VEGF expression promotes thyroid cancer cell growth, subsequent lymph node metastasis, local invasion, and distant metastasis, whereas the inhibition of VEGF signaling results in suppression of the tumor growth (Lin & Chao 2005).
A previous study reported that plasma VEGF levels in advanced thyroid cancer patients were significantly elevated (Lin et al. 2003). A 5-year cohort study showed that expression of VEGF was strongly associated with a higher frequency of local and distant recurrence of papillary thyroid carcinoma (Kilicarslan et al. 2003).
Some polymorphisms in the promoter and 3′-untranslated region are known to regulate VEGF expression (Brogan et al. 1999, Awata et al. 2002). Three single nucleotide polymorphisms (SNPs; −2578C/A, −634G/C in the promoter region and +936C/T in the 3′-untranslated region) are common in the Han Chinese population (http://www.ncbi.nlm.nih.gov/SNP/) and are related to VEGF protein production (Rogers & D’Amato 2006). The current SNP nomenclature for the above three SNPs is rs699947 for −2578C/A, rs2010963 for −634G/C, and rs3025039 for +936C/T. VEGF polymorphisms have been implicated in the susceptibility to several cancers including prostate cancer (Sfar et al. 2006), breast cancer (Kataoka et al. 2006), lung cancer (Koukourakis et al. 2004), gastric cancer (Tzanakis et al. 2006), and renal cell carcinoma (Kawai et al. 2007). However, the effect of these polymorphisms on thyroid cancer has not been reported. On the basis of the biological and pathologic significance of VEGF, it is possible that functional genetic variations in the VEGF gene may contribute to the development and progression of thyroid cancer. To test the hypothesis, we conducted a case–control study to evaluate the effects of these three functional polymorphisms on the development of thyroid cancer and lymph node metastasis.
Materials and Methods
Subjects
We initially recruited 332 patients with thyroid cancer (only PTC or FTC was enrolled) and 261 controls at the Kaohsiung Medical University Memorial Hospital in Taiwan. The initial diagnostic year of thyroid cancer was from 1980 to 2006. The diagnosis with thyroid cancer and lymph node metastasis were both confirmed by pathological examination. The presence of regional lymph node metastasis was detected while the patient receiving thyroidectomy. The control subjects were patients from the Department of Endocrinology and Otorhinolaryngology for other reasons. None of the controls had known thyroid disease or any type of cancer upon enrollment. All of the control subjects received thyroid physical examination by an endocrinologist or otolarygologists. Twenty-one control subjects with simple goiter have been further evaluated by thyroid sonography and fine needle aspiration to exclude any malignant change. The female versus male ratio was higher in the cases than controls. To reduce the impact of unbalanced sex ratio in this case–control study, we subsequently enrolled another 156 female controls and genotyped the ‘interesting’ SNP based on the results from the initial study subjects. These 156 female controls were recruited from the healthy individuals who came to the Department of Preventive Medicine for annual health checkup. None of them had reported cancer or thyroid disease. Information on demographic characteristics, family history of cancer, and exposure to ionizing radiation were collected. The study was proved by the Institutional Review Board of Kaohsiung Medical University Hospital and written informed consent was given by each subject or custodian (if the age of the participant was <18 years old).
Genotyping
Genomic DNA was extracted from peripheral blood by a standard method. Genotyping was carried out by the TaqMan technology. Briefly, PCR primers and TaqMan minor groove binder probes were designed and reactions were performed in 96-well microplates with ABI 9700 thermal cyclers (Applied Biosystems, Foster City, CA, USA). The information about probe sequences and PCR can be found in Supplementary Table 1 which can be viewed online at http://joe.endocrinology-journals.org/content/vol195/issue2/. Fluorescence was measured with an ABI 7500 Real-Time PCR System and analyzed with its System SDS software version 1.2.3.
Statistical analysis
SPSS for Windows 13.0 version was used for statistical analysis. Continuous variables were analyzed by independent t-test and were presented as mean±s.d. Allele frequencies were estimated by direct gene counting. Observed numbers of each genotype were compared with those expected for Hardy–Weinberg equilibrium (HWE) using the χ2-test. Genetic effects were first assessed by χ2-test or Fisher’s exact test. Multiple logistic regression analysis was performed to adjust for the effects of age and sex while assessing the genetic effects. A trend test (Ptrend) assuming a dose–response with increasing number of a risk allele was also performed. Haploview was applied to calculate linkage disequilibrium (LD) and haplotype blocks. Haplotype analysis was conducted using the Haclustering program (Tzeng et al. 2006). We used Bonferroni correction to account for multiple testing, and a two-tailed P value <0.017 (=0.05/3 SNPs) was considered statistically significant.
Results
Table 1 shows the baseline characteristics of the subjects. The genotyping call rate for each SNP ranged from 88 to 92%. The age of study group ranged from 11 to 82 years old. No significant difference of age was found between the case and control groups. Females were predominant among our cases (P<0.001), which is in agreement with the gender distribution in patients with thyroid cancer. Histological classification of thyroid tumor was as follows: 85.3% (255 patients) papillary cancer and 14.7% (44 patients) follicular cancer. Regional lymph node metastasis was detected in 26.4% (76 patients) of all thyroid cancer patients while receiving thyroidectomy. All the cases denied previous exposure to ionizing radiation sources (either accidental or therapeutic).
The distribution of VEGF genotypes was in HWE in either cases or controls. The minor A allele of rs699947 was over represented in the patients with lymph node metastasis than controls (33.6 vs 23.3%, P=0.014). However, the difference of A allele frequencies between all patients and controls was borderline significant (28.1% in cases vs 23.3% in controls, P=0.070; Table 2). The multivariate logistic regression model yielded an adjusted OR of 136 (95% CI= 1.02–1.81, P=0.039) for the risk A allele, while age and sex were adjusted for and an additive mode of inheritance was assumed. We also found that the frequency for either the AC or AA genotype was highest in patients with lymph node metastasis, followed by patients without lymph node metastasis, and lowest in controls. The proportion of the A allele carriers from these three types of subjects yielded a trend test P value of 0.019 (57.1 vs 45.0 vs 40.5%), which was close to Bonferroni-adjusted significance. When we analyzed each type of thyroid cancer, the results were less significant but the pattern of association was still present (Supplementary Tables 2 and 3 which can be viewed online at http://joe.endocrinology-journals.org/content/vol195/issue2/). For SNPs rs2010963 and rs3025039, we did not find any significant association between genotypes and phenotypes under dominant, recessive, or additive genetic model. When these two SNPs were analyzed for the patients with lymph nodes metastasis, neither of them reached to the statistical significance.
The sex distribution was very different between cases and controls. We genotyped SNP rs699947 in additional 156 female controls and found that the A allele frequency in the control increased from the original 23.3 to 24.7%. These controls did not change the potential effect of SNP rs699947. We further divided the original study subjects by sex and examined the sex-specific genetic effect (Table 3). We found that the association between SNP rs699947 and the risk of thyroid cancer existed only in men (adjusted OR=1.97, 95% CI=1.16–3.37, P=0.013) but not in women (adjusted OR=1.15, 95% CI= 0.81–1.62, P=0.435). The other two SNPs still did not show significant sex-specific effect. The two SNPs rs699947 and rs2010963 were in strong LD (D′=0.96), but both SNPs had low LD (D′<0.15) with the third SNP rs3025039. Haplotype analysis using the two SNPs in the same block demonstrated a less significant result (an empirical P value of 0.07).
Discussion
In the present study, we examined the relationship between three functional SNPs of the VEGF gene and the risk of thyroid cancer and regional lymph node involvement. Our results suggest a potential role of the VEGF promoter SNP rs699947 (i.e. −2578 C/A) in the development of thyroid cancer and lymph node metastasis. The A allele of SNP rs699947 increases a risk for thyroid cancer development, and may also facilitate lymph node metastasis. The effect of this risk allele is present in men but not in women. The association between genetic variation in VEGF and thyroid cancer is biologically plausible for several reasons: first, VEGF has been identified as a critical factor in angiogenesis required for tumor growth (Bunone et al. 1999). Second, VEGF expression has been related to tumor progression (Viglietto et al. 1995, Lewy-Trenda & Wierzchniewska-Lawska 2002) and metastasis (Fellmer et al. 1999, Klein et al. 1999). The promoter polymorphism of the VEGF gene in our study in part explains the variation of individual susceptibility to thyroid cancer and regional lymph node involvement. However, our positive result is based on a small number of male subjects. Further studies to replicate our result are necessary. Before any further replication, our result should be considered potentially intriguing but tentative.
It is of particular interest that the genetic risk of rs699947 only exists in males. Although the prevalence of thyroid cancer in women is in excess of men, being male is regarded as one of the high risk factors for thyroid cancer recurrence and cancer death (Liska et al. 2005). A growing body of evidence suggests that sex hormone plays a role in regulating VEGF expression. Studies showed that androgens can stimulate VEGF expression (Haggstrom et al. 1999, Stewart et al. 2001, Woodward et al. 2005). Androgens can activate hypoxia inducible factor-1 (Mabjeesh et al. 2003) which in turn translocates into nucleus to enhance VEGF expression (Josko & Mazurek 2004). On the other hand, studies also demonstrate that both estrogen and progestin can increase VEGF expression. Garvin et al.(2005) showed that estrogen acts as an angiogenic switch in breast cancer cells. Furthermore, progesterone-response elements had been identified in the promoter of the VEGF gene (Mueller et al. 2003). Mutation in the Sp-1 region of VEGF promoter can abolish progestin induction of VEGF synthesis (Wu et al. 2005). The role of sex hormone in regulation of VEGF expression in thyroid cancer has not been explored and our finding of sex-specific VEGF genetic effect has yet to be replicated by other studies.
Twenty-nine cases with genotypes at SNP rs699947 do not have information regarding lymph node metastasis. If all these 29 cases have a systemic bias towards the group with lymph node metastasis, the result is still significant after adding all of them into statistical models. On the other hand, if the systemic bias is towards the group without lymph node metastasis, the statistical result remains significant. For the patients without evidence of lymph node metastasis, it is possible that the surgeons did not remove the involved lymph nodes leading to an underestimation of the percentage of lymph node metastasis. However, this bias would be independent of genotypes and should not result in a different conclusion. Retrospectively recruiting post-operative patients could lead to survival bias. To test for this potential bias and its impact on our conclusion, we divided the subjects into two groups by the follow-up period ≥5 and <5 years. We found that the frequency of the risk A allele of SNP rs699947 was 28.0% in the long follow-up group and 28.5% in the short follow-up group. Therefore, the ascertainment of this retrospective case–control study did not have survival bias.
In conclusion, we found that the +2578 C/A SNP in the promoter region of the VEGF gene may predispose the risk of development of thyroid cancer and regional lymph node metastasis. Our data also suggested a sex-specific effect and males are under stronger genetic influence than females.
Baseline demographics of study characteristics
| Case (n=332) | Control (n=261) | P value | |
|---|---|---|---|
| aMissing information of histological type in 33 thyroid cancer patients. | |||
| bLymph node metastasis (+): papillary cancer, n=68; follicular cancer, n=8. | |||
| Age (mean±s.d., range, y/o) | 43.3±13.1 (11–82) | 42.6±15.0 (11–81) | 0.583 |
| Sex (male: female) | 1:4.75 | 1:1.24 | 0.001 |
| Pathologya | |||
| Papillary cancer | 255 (85.3%) | – | – |
| Follicular cancer | 44 (14.7%) | – | – |
| Lymph node metastasis | |||
| Yesb | 76 (26.4%) | – | – |
| No | 212 (73.6%) | – | – |
Association between vascular endothelial growth factor polymorphisms and thyroid cancer
| Thyroid cancer, n (%) | ||||||
|---|---|---|---|---|---|---|
| Total | LN meta (+) | LN meta (−) | Control, n (%) | χ2P value | Ptrend | |
| LN meta: lymph node metastasis. P1 and P1′: total thyroid cancer versus control. P2 and P2′ : thyroid cancer with LN meta (+) versus control. Trend test: genotypes versus LN meta (+)→LN meta (−)→control. | ||||||
| Genotype | ||||||
| rs699947(K2578C/A) | ||||||
| CC | 155 (52.2%) | 30 (42.9%) | 109 (55.0%) | 148 (59.5%) | P1=0.202 | 0.019 |
| AC | 117 (39.4%) | 33 (47.1%) | 73 (36.9%) | 86 (34.5%) | P2=0.043 | |
| AA | 25 (8.4%) | 7 (10.0%) | 16 (8.1%) | 15 (6.0%) | ||
| Allele | ||||||
| C | 427 (71.9%) | 93 (66.4%) | 291 (73.5%) | 382 (76.7%) | P1′ =0.070 | |
| A | 167 (28.1%) | 47 (33.6%) | 105 (26.5%) | 116 (23.3%) | P2′ =0.014 | |
| rs2010963(K634G/C) | ||||||
| GG | 104 (33.2%) | 20 (28.6%) | 68 (32.4%) | 67 (29.1%) | P1=0.438 | 0.454 |
| CG | 160 (51.1%) | 40 (57.1%) | 108 (51.4%) | 119 (51.7%) | P2=0.606 | |
| CC | 49 (15.7%) | 10 (14.3%) | 34 (16.2%) | 44 (19.1%) | ||
| Allele | ||||||
| G | 368 (58.8%) | 80 (57.1%) | 244 (58.1%) | 253 (55.0%) | P1′ =0.213 | |
| C | 258 (41.2%) | 60 (42.9%) | 176 (41.9%) | 207 (45.0%) | P2′ =0.655 | |
| rs3025039(C936C/T) | ||||||
| CC | 171 (59.4%) | 43 (65.2%) | 101 (55.2%) | 145 (62.5%) | P1=0.739 | 0.867 |
| CT | 105 (36.5%) | 21 (31.8%) | 73 (39.9%) | 77 (33.2%) | P2=0.862 | |
| TT | 12 (4.2%) | 2 (3.0%) | 9 (4.9%) | 10 (4.3%) | ||
| Allele | ||||||
| C | 447 (77.6%) | 107 (81.1%) | 275 (75.1%) | 367 (79.1%) | P1′ =0.562 | |
| T | 129 (22.4%) | 25 (18.9%) | 91 (24.9%) | 97 (20.9%) | P2′ =0.621 | |
Sex-specific association between rs699947(+2578C/A) and thyroid cancer
| Thyroid cancer, n (%) | |||||||
|---|---|---|---|---|---|---|---|
| Total | LN meta (+) | LN meta (−) | Control, n (%) | Adjusted ORa (95% CI) | Sig. | Ptrendb | |
| aComparing total cases with controls by multiple logistic regression where age was adjusted and an additive mode of inheritance was assumed. | |||||||
| bPtrend: genotypes in LN meta (+)→LN meta (−)→control. | |||||||
| Genotype | |||||||
| Male | |||||||
| CC | 23 (41.1%) | 7 (30.4%) | 11 (42.3%) | 70 (62.5%) | 1.97 | P=0.013 | 0.002 |
| AC+AA | 33 (58.9%) | 16 (69.6%) | 15 (57.7%) | 42 (37.5%) | (1.16–3.37) | ||
| Female | |||||||
| CC | 132 (55.0%) | 23 (48.9%) | 98 (57.0%) | 77 (56.6%) | 1.15 | P=0.435 | 0.489 |
| AC+AA | 108 (45.0%) | 24 (51.1%) | 74 (43.0%) | 59 (43.4%) | (0.81–1.62) | ||
(P-J Hsiao and M-Y Lu contributed equally to this work)
This work was supported by the grants from the National Science Council, Taiwan NSC 94–2314B037–104, and from the Cancer Research Foundation of Kaohsiung Medical University (QC-094006 and QC-094001). We are grateful for Dr Yi-Chu Liao for statistical analysis and Wansu Chang for technical assistance. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Akslen LA & Livolsi VA 2000 Increased angiogenesis in papillary thyroid carcinoma but lack of prognostic importance. Human Pathology 31 439–442.
Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M, Inukai K, Inoue I & Katayama S 2002 A common polymorphism in the 5′-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes 51 1635–1639.
Brogan IJ, Khan N, Isaac K, Hutchinson JA, Pravica V & Hutchinson IV 1999 Novel polymorphisms in the promoter and 5′ UTR regions of the human vascular endothelial growth factor gene. Human Immunology 60 1245–1249.
Bunone G, Vigneri P, Mariani L, Buto S, Collini P, Pilotti S, Pierotti MA & Bongarzone I 1999 Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features. American Journal of Pathology 155 1967–1976.
Carmeliet P & Jain RK 2000 Angiogenesis in cancer and other diseases. Nature 407 249–257.
DeLellis RA, Lloyd RV & Heitz PU 2004 Pathology and genetics of tumor endocrine organs, World Health Organization Classification of Tumors. International Agency for Research on Cancer 8 57–66.
Fellmer PT, Sato K, Tanaka R, Okamoto T, Kato Y, Kobayashi M, Shibuya M & Obara T 1999 Vascular endothelial growth factor-C gene expression in papillary and follicular thyroid carcinomas. Surgery 126 1056–1061 (discussion 1061–1052).
Garvin S, Nilsson UW & Dabrosin C 2005 Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells. British Journal of Cancer 93 1005–1010.
Haggstrom S, Lissbrant IF, Bergh A & Damber JE 1999 Testosterone induces vascular endothelial growth factor synthesis in the ventral prostate in castrated rats. Journal of Urology 161 1620–1625.
Josko J & Mazurek M 2004 Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis. Medical Science Monitor 10 RA89–RA98.
Kataoka N, Cai Q, Wen W, Shu XO, Jin F, Gao YT & Zheng W 2006 Population-based case–control study of VEGF gene polymorphisms and breast cancer risk among Chinese women. Cancer Epidemiology, Biomarkers and Prevention 15 1148–1152.
Kawai Y, Sakano S, Korenaga Y, Eguchi S & Naito K 2007 Associations of single nucleotide polymorphisms in the vascular endothelial growth factor gene with the characteristics and prognosis of renal cell carcinomas.European Urology. In press.
Kilicarslan AB, Ogus M, Arici C, Pestereli HE, Cakir M & Karpuzoglu G 2003 Clinical importance of vascular endothelial growth factor (VEGF) for papillary thyroid carcinomas. Acta Pathologica, Microbiologica et Immunologica Scandinava 111 439–443.
Klein M, Picard E, Vignaud JM, Marie B, Bresler L, Toussaint B, Weryha G, Duprez A & Leclere J 1999 Vascular endothelial growth factor gene and protein: strong expression in thyroiditis and thyroid carcinoma. Journal of Endocrinology 161 41–49.
Kondo T, Ezzat S & Asa SL 2006 Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nature Reviews. Cancer 6 292–306.
Koukourakis MI, Papazoglou D, Giatromanolaki A, Bougioukas G, Maltezos E & Sivridis E 2004 VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer. Lung Cancer 46 293–298.
Lewy-Trenda I & Wierzchniewska-Lawska A 2002 Expression of vascular endothelial growth factor (VEGF) in human thyroid tumors. Polish Journal of Pathology 53 129–132.
Lin JD & Chao TC 2005 Vascular endothelial growth factor in thyroid cancers. Cancer Biotherapy and Radiopharmaceuticals 20 648–661.
Lin SY, Wang YY & Sheu WH 2003 Preoperative plasma concentrations of vascular endothelial growth factor and matrix metalloproteinase 9 are associated with stage progression in papillary thyroid cancer. Clinical Endocrinology 58 513–518.
Liska J, Altanerova V, Galbavy S, Stvrtina S & Brtko J 2005 Thyroid tumors: histological classification and genetic factors involved in the development of thyroid cancer. Endocrine Regulations 39 73–83.
Mabjeesh NJ, Willard MT, Frederickson CE, Zhong H & Simons JW 2003 Androgens stimulate hypoxia-inducible factor 1 activation via autocrine loop of tyrosine kinase receptor/phosphatidylinositol 3′-kinase/protein kinase B in prostate cancer cells. Clinical Cancer Research 9 2416–2425.
Malchoff CD & Malchoff DM 2006 Familial nonmedullary thyroid carcinoma. Cancer Control 13 106–110.
Mueller MD, Vigne JL, Pritts EA, Chao V, Dreher E & Taylor RN 2003 Progestins activate vascular endothelial growth factor gene transcription in endometrial adenocarcinoma cells. Fertility and Sterility 79 386–392.
Rogers MS & D’Amato RJ 2006 The effect of genetic diversity on angiogenesis. Experimental Cell Research 312 561–574.
Schlumberger MJ & Torlantano M 2000 Papillary and follicular thyroid carcinoma. Baillieres of Best Practice and Research. Clinical Endocrinology and Metabolism 14 601–613.
Segal K, Shpitzer T, Feinmesser M, Stern Y & Feinmesser R 1996 Angiogenesis in follicular tumors of the thyroid. Journal of Surgical Oncology 63 95–98.
Sfar S, Hassen E, Saad H, Mosbah F & Chouchane L 2006 Association of VEGF genetic polymorphisms with prostate carcinoma risk and clinical outcome. Cytokine 35 21–28.
Stewart RJ, Panigrahy D, Flynn E & Folkman J 2001 Vascular endothelial growth factor expression and tumor angiogenesis are regulated by androgens in hormone responsive human prostate carcinoma: evidence for androgen dependent destabilization of vascular endothelial growth factor transcripts. Journal of Urology 165 688–693.
Turner HE, Harris AL, Melmed S & Wass JA 2003 Angiogenesis in endocrine tumors. Endocrine Reviews 24 600–632.
Tzanakis N, Gazouli M, Rallis G, Giannopoulos G, Papaconstantinou I, Theodoropoulos G, Pikoulis E, Tsigris C, Karakitsos P, Peros G et al.2006 Vascular endothelial growth factor polymorphisms in gastric cancer development, prognosis, and survival. Journal of Surgical Oncology 94 624–630.
Tzeng JY, Wang CH, Kao JT & Hsiao CK 2006 Regression-based association analysis with clustered haplotypes through use of genotypes. American Journal of Human Genetics 78 231–242.
Viglietto G, Maglione D, Rambaldi M, Cerutti J, Romano A, Trapasso F, Fedele M, Ippolito P, Chiappetta G, Botti G et al.1995 Upregulation of vascular endothelial growth factor (VEGF) and downregulation of placenta growth factor (PlGF) associated with malignancy in human thyroid tumors and cell lines. Oncogene 11 1569–1579.
Woodward WA, Wachsberger P, Burd R & Dicker AP 2005 Effects of androgen suppression and radiation on prostate cancer suggest a role for angiogenesis blockade. Prostate Cancer and Prostatic Diseases 8 127–132.
Wu J, Brandt S & Hyder SM 2005 Ligand- and cell-specific effects of signal transduction pathway inhibitors on progestin-induced vascular endothelial growth factor levels in human breast cancer cells. Molecular Endocrinology 19 312–326.
