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.

Abstract Of the 44,670 new cases of thyroid cancer predicted for this year in the US by the American Cancer Society, 80% will be papillary thyroid carcinoma (PTC). PTC typically arises from a gain-of-function mutation in the RET, RAS, or BRAF genes which make up a linear signaling cascade for ERK activation in over 90% of PTC cases. PTCs possessing the BRAFV600E mutation have been associated with greater local invasion and regional metastatic potential. Urokinase plasminogen activator (uPA) has been shown to be an important mediator of invasion and metastasis in several cancers. To determine the extent to which BRAFV600E mutation status predicts up-regulation of the uPA in PTC, a study using patient thyroid tissue samples obtained over a 6-year period at a single tertiary care center was undertaken. DNA and RNA were obtained from patient PTC and matched, normal thyroid tissue samples using the Trizol method. BRAFV600E mutational status of the DNA was determined using the TaqMan SNP genotyping assay, while RNA was analyzed for differences in uPA transcription levels (relative to matched, normal thyroid tissue) by qRT-PCR. Fifty-four percent of the PTC samples possessed the BRAFV600E mutation. PTC samples bearing the BRAFV600E mutation displayed significantly higher uPA RNA levels (relative to matched control tissue) than those samples with wild-type BRAF (5.883 vs. 1.27-fold, p<0.05, Wilcoxon signed-rank test). Additionally, uPA RNA levels were significantly higher in patients with nodal metastasis (p<0.05). These data provide new evidence of the roles of BRAFV600E and uPA in PTC invasive pathology and demonstrate for the first time that BRAFV600E status is able to predict higher uPA levels in PTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2011-1063

Abstract In the last decade, the incidence of thyroid cancer has risen ∼84% and mortality ∼34%. The ACS estimates ∼ 37,200 new cases of thyroid cancer, with 1,630 deaths in 2009. Papillary thyroid carcinoma (PTC) is the most common thyroid and endocrine malignancy, accounting for ∼80% of all thyroid cancer. It typically occurs from a gain-of-function mutation in the RET, RAS or BRAF genes which make up a linear signaling cascade for ERK activation. Recurrent or persistent disease prevails in up to 40% of thyroidectomy cases with a poor prognosis when aggressive cancer is evident. Thus, further research into factors contributing to aggressive disease is merited. Matrix metalloproteinases (MMPs) represent a family of secreted proteases with diverse functions implicated in the pathogeneses of aggressive tumors. Their functions include degrading the extracellular matrix (ECM) to facilitate cell migration, cell signaling through ECM cleavage products that can affect apoptosis and cell growth, and they can act directly on growth factor precursors and receptors. MMP upregulation has been demonstrated in many types of cancers yet its pattern of expression is poorly understood in PTC. Analysis of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the human PTC cell line, BCPAP, and the normal thyroid cell line, NTHY-ori by protein array and real-time RT-PCR revealed significantly elevated levels of MMPs and decreased levels of TIMP-4. Real time RT-PCR demonstrated the following fold increases in expression in BCPAP compared to NTHY-ori: 16. 9 (SE=0.79) for MMP-1, 1.80 (SE=0.41) for MMP-3, 1.40 (SE=0.08) for MMP-9, 7.12 (SE=2.14) for MMP-10, 12.60 (SE=6.90) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 2.38 (SE=1.04). Protein array data from serum free conditioned media of the cell lines reflected a similar pattern of secreted MMP and TIM4 levels. Fold increases in the optical concentration (determined by spot densitometry) of secreted proteins were detected in BCPAP compared to NTHY-ori of 29.48 (SE=1.92) for MMP-1, 16.80 (SE=1.07) for MMP-3, 1.66 (SE=0.07) for MMP-9, 3.66 (SE=0.55) for MMP-10, 1.59 (SE=0.11) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 1.27 (SE=0.06). For the first time, these results demonstrate increased production and secretion of a group of MMPs and down regulation of TIMP-4 which have been characterized in tumor progression in a PTC cell line. Further investigation into the role these agents play in the tumorgenesis of aggressive PTC may yield insight into future treatment and screening alternatives. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 529.

Abstract Papillary thyroid cancer (PTC) is the most prevalent endocrine and thyroid malignancy. The ability of PTC to invade and migrate great distances contributes to distant metastases, which represent the most common cause of PTC-related death. The urokinase plasminogen activator (uPA) and the urokinase plasminogen receptor (uPAR) are key mediators of tumor invasion. Upon binding to uPAR, pro-uPA is converted to its active form, which is then capable of cleaving plasminogen to plasmin. Plasmin can then degrade components of the basement membrane and extracellular matrix, a prerequisite for tumor cell invasion and metastasis. The binding of uPA by uPAR also seems to mediate several signaling events that contribute to a migratory phenotype, as well as various growth signals. In this study, we analyzed uPA and uPAR expression in PTC and normal thyroid tissue, and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, as well as the functional consequences of inhibiting this system. 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 BCPAP cells compared to NTHY-Ori-3-1. Fluorimetric assays revealed that BCPAP CM was able to activate plasminogen, resulting in measureable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. The in vitro invasion phenotype of BCPAP cells was augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. Cells transfected with siRNA against uPAR demonstrated decreased migratory and invasive potentials as measured by Matrigel invasion assays. Finally, uPAR-knockdown cells exhibited decreased proliferation when compared to non-targeting siRNA transfectant cells. 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 520.

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.

Proc Amer Assoc Cancer Res, Volume 47, 2006 3481 Thyroid cancer has become the most prevalent endocrine malignancy in the US. In the last decade, the incidence has risen 84% and mortality 34% thus making it a significant clinical concern, meriting research in the areas of treatment, detection and prevention. Both clinical and animal studies have correlated high dietary fat intake with an increased incidence of cancer in various tissues. More specifically, studies have implicated polyunsaturated ω-6 fatty acids such as linoleic and arachidonic acids with increased tumor growth. Recent studies show increased leukotriene synthesis, particularly the potent inflammatory chemokines in the 5-lipoxygenase pathway, with the promotion of certain cancers, including breast, lung and prostate. These studies also demonstrated the potential therapeutic effects of lipoxygenase inhibitors in cancer treatment. Additionally, the detection of 15-lipoxygenase in lung cancer is being investigated as a potential prognostic marker. Expression analysis of 7 papillary thyroid tumors vs. paired, normal thyroid tissue, revealed an approximately 5 fold increased in expression of both 5-lox (p = 0.004) and 15-lox-2 (p = 0.09) in all 7 patients. Subsequent down stream enzymes were also elevated in these patients. Thus far, 5-lox expression was validated by real-timer RT-PCR. Increased protein expression of both for 5-lox and 15-lox-2 were observed by western blotting. This is the first time, to our knowledge, that increased activity in the metabolism of arachidonic acid pathway has been demonstrated in papillary thyroid carcinoma, implicating lipoxygenases as a potential therapeutic target and prognostic marker. In addition these data suggest dietary considerations in at risk individuals and patients.