Pigment epithelium‑derived factor has a role in the progression of papillary thyroid carcinoma by affecting the HIF1α‑VEGF signaling pathway
- Authors:
- Published online on: October 25, 2016 https://doi.org/10.3892/ol.2016.5316
- Pages: 5217-5222
Abstract
Introduction
Thyroid cancer is the most common endocrine malignancy and papillary thyroid carcinoma (PTC) accounts for 80–90% of all thyroid malignancies (1,2). The incidence of PTC has increased in most countries in the last three decades (3,4). Although the majority of patients with PTC have a favorable prognosis following appropriate treatments, 10–30% of patients with PTC progress to metastasis or recurrence, and mortality in 5% of cases (5,6). The mechanisms underlying the progression of PTC have yet to be elucidated.
For the majority of patients with PTC, metastasis remains the main cause of mortality; >90% of cancer-associated mortalities are caused by tumor invasion and metastasis, which emphasizes the importance of elucidating the mechanisms underlying metastasis (7–9). The contribution of angiogenesis to tumor progression is well established. In human solid cancer, the growth and survival of tumor cells are angiogenesis-dependent. Following the inhibition of angiogenesis, metastasis of the primary tumor is affected (10,11). Therefore, it is necessary to investigate novel factors that may inhibit metastasis by affecting angiogenesis in human PTC.
Pigment epithelium-derived factor (PEDF) is a 50-kDa secreted glycoprotein that was initially identified in cultured retinal pigment epithelial cells (12). PEDF has numerous biological functions, including differentiating activity, neurite outgrowth, survival activity, anti-apoptosis and anti-angiogenic activities and the induction of cell death (13–19). The anticancer role of PEDF has yet to be elucidated; however, previous studies have suggested various roles for PEDF in inhibiting cancer progression. For instance, PEDF may cause tumors to differentiate to a less malignant phenotype, PEDF may block angiogenesis-mediated activities and neovascularization, and PEDF may suppress tumor cell invasion and metastasis (14,15).
PEDF is one of the most promising anti-angiogenic factors; a number of studies have demonstrated its anti-angiogenic effects in various tumor models, including retinoblastoma, neuroblastoma, prostate cancer, melanoma, Wilms' tumor, pancreatic adenocarcinoma, hepatoblastoma, osteosarcoma, chondrosarcoma, human cervical carcinoma, gastric carcinoma, nasopharyngeal carcinoma, Lewis lung carcinoma, colorectal peritoneal carcinoma, glioma and breast cancer xenografts (20–24). The anti-angiogenic effect of PEDF is performed primarily through the disruption of microvascular network distribution (25–28). Vascular endothelial growth factor (VEGF) is an established pro-angiogenic factor and numerous studies have reported an inverse correlation between PEDF and VEGF expression levels in certain tumor models (21,25–28).
The degree of oxygenation is crucial to neovascularization (29). The transcription factor hypoxia-inducible factor 1α (HIF1α) is a critical protein involved in the response to hypoxia, and is able to activate several downstream factors, including VEGF and glucose transporter 1; its expression is associated with tumor progression in various carcinomas, including pancreatic cancer, breast cancer, cervical carcinoma and thyroid cancer (29–31). Considering the associations between PEDF and tumor progression, PEDF expression levels in patients with PTC were evaluated in the current study. To the best of our knowledge, the present study is the first to investigate the association between PEDF expression levels and aggressive clinicopathological features in PTC, and to determine whether PEDF affects the lymph node metastasis (LNM) process in PTC by altering the HIF1α-VEGF signaling pathway.
Materials and methods
Patients
Patients with PTC who underwent thyroid excision surgery during the period 2011–2013 at The Second Affiliated Hospital of Harbin Medical University (Harbin, China) were recruited for the present study. The standard pathological diagnosis of PTC was based on the World Health Organization criteria (32) and two pathologists independently reviewed histological specimens in a blinded manner. A total of 271 patients with PTC (24 males and 247 females) were diagnosed and included in the current study. All patients met the inclusion criteria, which were: i) Underwent thyroid excision surgery; ii) confirmed as PTC by intraoperative rapid pathology and postoperative pathological detection; and iii) no history of thyroid disease and thyroid-related medication use. Informed consent was obtained from all participants and the study was performed according to the guidelines of the Ethics Committee of the Harbin Medical University.
Relevant patient data were collected, including demographic features (gender and age), clinical features (thyroid-stimulating hormone levels, tumor size and Hashimoto's disease) and pathological features (multifocality, T stage, extrathyroid invasion, intact capsule, prophylactic central compartment lymph-node (neck) dissection and node metastases). Tumor-node-metastasis (TNM) classification was performed according to the American Joint Committee on Cancer (33). T1 was defined as a tumor ≤2 cm in diameter and limited to the thyroid gland. T2 was defined as a tumor that was >2 and ≤4 cm, and limited to the thyroid gland. T3 was defined as a tumor >4 cm and limited to the thyroid gland, or any tumor with minimal glandular infiltration, including infiltration of the sternothyroid muscle or perithyroid soft tissues.
Immunohistochemical analysis
PTC tissue specimens were sectioned (4-µm), deparaffinized in xylene and rehydrated in a graded series of ethanol. The slides were then incubated in 3% hydrogen peroxide in distilled water at room temperature for 10 min to inactivate endogenous peroxidase activity, and subsequently underwent antigen retrieval in a sodium citrate solution in a microwave oven. The tissue sections were blocked with 5% bovine serum albumin (Boster Bio-Engineering, Wuhan, China) for 30 min at room temperature, and then incubated overnight at 4°C with a rabbit anti-human PEDF primary polyclonal antibody at 1:100 dilution (#BA1348-1; Boster Bio-Engineering) in a humidified chamber. The tissue sections were then incubated with a ready-to-use biotinylated horseradish-peroxidase-conjugated secondary antibody (#SA1022; Boster Bio-Engineering) and a streptavidin biotin complex (#SA1022; Boster Bio-Engineering) at 37°C for 30 min. The staining procedures were performed according to the manufacturer's instructions: Visualization with a 3,3′-diaminobenzidine solution and counterstaining with hematoxylin. The distribution and expression levels of PEDF were examined from the images obtained using the Olympus Imaging system (DP73: Olympus Corporation, Tokyo, Japan).
PEDF expression levels were semi quantitatively categorized into three groups, as follows: Negative (0 points), ≤5% positive cells; weakly positive (1 point), 6–30% positive cells; moderately positive (2 points), 31–60% positive cells.
Laser-capture microdissection
The frozen tissues specimens obtained from PTC patients with LNM (n=15) and without LNM (n=10) were used for laser capture microdissection (ArcturusXT™; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to obtain target thyroid epithelial cells, as described previously (34). The age and gender of the participants were matched for each group.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the microdissected cells using RNAiso Plus (9108; Takara Biotechnology Co., Ltd., Dalian, China). The extracted RNA was subsequently incubated with Recombinant DNase I (D2270; Takara Biotechnology Co., Ltd.) to erase the genomic DNA. cDNA was then obtained from mRNA using the PrimeScript™ RT reagent kit (DRR025A; Takara Biotechnology Co., Ltd.), which was then amplified using PCR on the ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) with SYBR Green I dye (DRR041S; Takara Biotechnology Co., Ltd.), in accordance with the manufacturer's protocol. The cycling conditions were as follows: 1 cycle as an initial denaturation at 95°C for 10 min; 40 cycles of 95°C for 5 sec, 60°C for 30 sec and 72°C for 15 sec; and a final extension step at 72°C for 5 min. The relative expression levels of PEDF, VEGF and HIF1α were determined using the comparative Cq method (35), following normalization to the endogenous GAPDH control gene. The primer sequences used in the RT-qPCR are presented in Table I.
Statistical analysis
All statistical analyses in the current study were performed with SPSS software, version 13.01S (SPSS, Inc., Chicago, IL, USA). An independent-sample t-test was used to compare the means between two groups, and a χ2 or Fisher's exact test was used to compare frequencies between the groups. The data are presented as the mean ± standard deviation, or as percentages where appropriate. A Pearson correlation analysis was used to analyze the associations among the expression levels of PEDF, VEGF and HIF1α in PTC cells. All t-tests were two-tailed; P<0.05 was considered to indicate a statistically significant difference.
Results
Clinicopathological characteristics of patients with PTC
The clinicopathological characteristics of the patients are shown in Table II. Between 2011 and 2013, 271 patients with PTC, including 24 male and 247 female patients, were enrolled in the present study. The mean age of the patients was 43.1±10.6 years (age range, 19–73 years) and the mean size of the tumor was 10.9±7.8 mm (range, 2–50 mm). Multifocal tumors were identified in 12 patients; 14 patients (5.2%) had extrathyroid invasion and 86 patients (31.7%) had LNM. Hashimoto's disease was observed in 45 patients. A total of 245 patients had T1 tumors, 26 had T3 tumors and none had tumors in another stage. The BRAFV600E mutation was present in 70.1% of patients. Immunohistochemistry with an anti-PEDF antibody detected PEDF expression in 74.5% of the PTC tissues. PEDF expression was determined to be negative in 69 patients, weakly positive in 131 patients and moderately positive in 71 patients.
 | Table II.Clinicopathological characteristics of patients with papillary thyroid carcinoma, recruited between 2011 and 2013. |
Decreased PEDF levels correlate with the progression of human PTC
In order to determine whether PEDF contributes to the progression of PTC, the associations between PEDF and aggressive clinicopathological features were investigated. As a result of the importance of metastasis to the progression of PTC, the association between PEDF expression levels and LNM was initially evaluated. As presented in Fig. 1 and Table III, PEDF expression levels were significantly decreased in PTC tissues with LNM, as compared with PTC tissues without LNM (P=0.006). Similarly, PEDF expression levels were significantly decreased in PTC tissues with extrathyroid invasion (P=0.035), a high TNM stage (P=0.013), BRAFV600E mutation positivity (P<0.001) and a tumor size of >10 mm (P=0.018). However, PEDF expression levels were not significantly associated with multifocality (P=0.152; Table III).
Decreased expression levels of PEDF are accompanied by an upregulation of the HIF1α-VEGF signaling pathway in patients with PTC and LNM
To elucidate the potential mechanisms underlying the role of PEDF in metastasis by affecting angiogenesis, the mRNA expression profiles of PEDF, VEGF and HIF1α in stored patient tissue samples were determined using RT-qPCR. As indicated in Fig. 2, PEDF mRNA expression levels were significantly decreased in PTC tissues with LNM (0.2766±0.0910), as compared with PTC tissues without LNM (0.6251±0.2034; P<0.01). By contrast, the mRNA expression levels of HIF1α and VEGF were significantly increased in PTC tissues with LNM (1.6646±0.5533 and 3.9321±0.9235, respectively), as compared with PTC tissues without LNM (0.6847±0.2240 and 0.7537±0.1988, respectively; P<0.01). In addition, as shown in Table IV, through the analysis of the mRNA expression levels of PEDF, VEGF and HIF1α in the thyroid tissues specimens obtained from PTC patients with LNM (n=15) and without LNM (n=10), the present study identified a significant inverse correlation between PEDF and VEGF expression levels (r=−0.514; P=0.009), an inverse association between PEDF and HIF1α expression levels (r=−0.287; P=0.164) and a significant positive correlation between VEGF and HIF1α expression levels (r=0.489; P=0.013).
Discussion
To the best of our knowledge, the present study is the first to demonstrate that PEDF expression levels are correlated with specific patient clinicopathological features, including LNM, extrathyroid invasion, BRAFV600E mutation positivity, tumor size and a high TNM stage in PTC tissues. The results also indicated that PEDF may have an important role in metastasis by affecting angiogenesis induced by HIF1α.
The actions of PEDF have been evaluated in various types of tumor cells. The results of a number of previous studies support the hypothesis that PEDF expression may promote tumor growth, invasion and metastasis (19,21,22,25,26). Reduced PEDF expression is a potent promoter of tumor growth and angiogenesis in breast cancer (36), and the loss of PEDF enables melanoma cells to acquire an invasive phenotype (37). The results of the present study were concordant with previous findings in the majority of solid tumors (22,26,36,37), which suggested that PEDF may have an important role in the progression of PTC. However, certain observations of the current study, including the association of PEDF expression levels with tumor stage and its lack of association with multifocality, must be investigated in further studies with a larger cohort, as only 26 cases of a T3 tumor and 12 cases of multifocality were recruited for the present study.
Considering its potent function in angiogenesis, it was hypothesized that PEDF exerts its role in metastasis mainly via affecting angiogenesis in PTC tissues. Previous studies have demonstrated that the HIF1α-VEGF signaling pathway is activated during the progression of thyroid cancer (38,39). To the best of our knowledge, the present study is the first to identify an inverse association between PEDF expression levels and the HIF1α-VEGF signaling pathway in human PTC tissues. Recent studies supported these findings. For instance, PEDF was able to suppress angiogenesis in a gastric carcinoma xenograft model by down regulating HIF1α and VEGF expression (27). Furthermore, PEDF suppressed tumor growth in a cervical cancer cell line by down regulating the expression of VEGF and HIF1α, which demonstrated the anti-angiogenic activity of PEDF (26). In addition, the HIF1α and VEGF/PEDF signaling pathway is targeted in impacting nasopharyngeal carcinoma cell proliferation and angiogenesis (28). PEDF may not be directly regulated by HIF1α, as indicated by the inverse association between PEDF and HIF1α identified in the current study. Therefore, HIF1α may also regulate the expression of PEDF via other factors in human PTC, which requires further analysis.
In conclusion, the results of the current study suggested that PEDF expression levels were significantly associated with certain aggressive clinicopathological features in human PTC, and indicated that PEDF may exert an anti-angiogenesis role by modulating the HIF1α-VEGF signaling pathway, which has an important role in the metastasis of PTC. Additional studies should further investigate these results using in vitro and in vivo models, and elucidate the mechanism underlying the function of PEDF in anti-angiogenesis, through the knock down of the PEDF gene.
Acknowledgements
This study was supported by the Natural Science Foundation of Heilongjiang Province of China (grant no. D201228).
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