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Background: Thyroid cancer is the most common endocrine malignancy worldwide, with the predominant form papillary thyroid carcinoma (PTC) representing approximately 80% of cases., Objective: This study was addressed to identify potential genes and pathways involved in the pathogenesis of PTC and potential novel biomarkers for this disease., Methods: Gene expression profiling was carried out by DNA microarray technology. Validation of microarray data by qRT-PCR, western blot, and enzyme linked immunosorbent assay was also performed in a selected set of genes and gene products, with the potential to be used as diagnostic or prognostic biomarkers, such as those associated with cell adhesion, extracellular matrix (ECM) remodeling and immune/inflammatory response., Results: In this study we found that upregulation of extracellular activities, such as proteoglycans, ECM-receptor interaction, and cell adhesion molecules, were the most prominent feature of PTC. Significantly over-expressed genes included SDC1 (syndecan 1), SDC4 (syndecan 4), KLK7 (kallikrein-related peptidase 7), KLK10 (kallikrein-related peptidase 10), SLPI (secretory leukocyte peptidase inhibitor), GDF15 (growth/differentiation factor-15), ALOX5 (arachidonate 5-lipoxygenase), SFRP2 (secreted Frizzled-related protein 2), among others. Further, elevated KLK10 levels were detected in patients with PTC. Many of these genes belong to KEGG pathway "Proteoglycans in cancer"., Conclusions: Using DNA microarray analysis allowed the identification of genes and pathways with known important roles in malignant transformation, and also the discovery of novel genes that may be potential biomarkers for PTC.

Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC., (Copyright © 2012 Wiley Periodicals, Inc.)

Objectives: To explore the induction of a proangiogenic phenotype in endothelial cells in the thyroid tumor microenvironment by estrogen-treated thyroid cancer cells and to define the role of vascular endothelial growth factor (VEGF) in this interaction., Design: Cell-based in vitro systems analysis., Subjects: Thyroid tumor cell lines (BCPAP [papillary thyroid cancer] and ML-1 [follicular thyroid cancer]) were cultured with estradiol with and without an estrogen receptor (ER) inhibitor (fulvestrant or ICI) and used to treat human umbilical vein endothelial cells (HUVECs)., Interventions: Immunofluorescence was used to confirm the presence of ERα and ERβ in BCPAP cells. Conditioned medium was then used to evaluate the induction of HUVEC tubulogenesis and migration. Secretion of VEGF in this medium was evaluated by Western blot analysis. The expression of phosphoinositide 3-kinase (PI3K), the initiator of a proangiogenic pathway, was evaluated with Western blot analysis of HUVEC lysates. The subsequent effects of an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody were also observed., Results: Estrogen receptor α and ERβ are expressed in thyroid cancer cells. Estrogen-stimulated ML-1 cells secreted an increased amount of VEGF likely as a result of ER signaling. In contact with this environment, HUVECs demonstrate enhanced tubulogenesis and migration. Western blot analysis documented estrogen-mediated upregulation of PI3K in HUVECs. These effects were mitigated by an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody., Conclusions: Our data provide evidence that estrogen can induce a proangiogenic endothelial cell phenotype in the thyroid tumor microenvironment through ER and VEGF signaling. Our findings suggest that the effect of antiestrogenic therapy targeting tumor angiogenesis can be enhanced through VEGF inhibition.

Papillary thyroid carcinoma (PTC) is the most common endocrine and thyroid malignancy.  The urokinase plasminogen activator receptor (uPAR) plays an important role in cancer pathogenesis, including breakdown of the extracellular matrix, invasion, and metastasis.  Additionally, there is increasing evidence that uPAR also promotes tumorigenesis via the modulation of multiple signaling pathways.  BRAFV600E, the most common initial genetic mutation in PTC, leads to ERK1/2 hyperphosphorylation, which has been shown in numerous cancers to induce uPAR.  Treatment of the BRAFV600E-positive PTC cell line, BCPAP, with the MEK/ERK inhibitor U0126 reduced uPAR RNA levels by 90%.  siRNA-mediated down-regulation of uPAR in BCPAP cells resulted in greatly decreased activity in the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.  This phenomenon was concurrent with drastically reduced proliferation rates and decreased clonigenic survival, as well as demonstrated senescence-associated nuclear morphology and induction of b-galactosidase activity. uPAR-knockdown BCPAP cells also displayed greatly reduced migration and invasion rates, as well as a complete loss of the cells' ability to augment their invasiveness following plasminogen supplementation. Taken together, these data provide new evidence of a novel role for uPAR induction (as a consequence of constitutive ERK1/2 activation) as a central component in PTC pathogenesis, and highlight the potential of uPAR as a therapeutic target.

Objectives/hypothesis: We analyzed the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in papillary thyroid carcinoma (PTC) and normal thyroid tissue and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, and the functional consequences of inhibiting this system., Study Design: Retrospective chart review of PTC patients, followed by prospective study using previously obtained patient tissue and PTC cellular models., Methods: uPA and uPAR RNA and protein levels were analyzed in PTC patient tissue samples, PTC and normal thyroid tissue culture cells, and conditioned media (CM) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and/or Western blotting. The plasminogen-activating ability of CM was examined using dark-quenched casein fluorimetry and casein-plasminogen gel zymography. The invasive potentials of the PTC and normal thyroid epithelial cell lines were assessed using an in vitro cellular invasion/migration system., Results: uPA and uPAR RNA and protein levels were increased in PTC patient samples and PTC cells relative to controls. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and Western blotting demonstrated increased active uPA secreted by PTC cells compared with normal thyroid cells. Fluorimetric assays revealed that the PTC cells' CM was able to activate plasminogen, resulting in measurable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. Finally, the in vitro invasion phenotypes of PTC cells were augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies., Conclusions: These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.