Androgen receptor activation decreases proliferation in thyroid cancer cells.

The American Cancer Society predicted more than 52 000 new cases of thyroid cancer in 2020, making it the most prevalent endocrine malignancy. Due to the approximately threefold higher incidence of thyroid cancer in women, we hypothesize that androgens and/or androgen receptors play a protective role and that thyroid cancer in men represents an escape from androgen-mediated cell regulation. The analysis of androgen receptor (AR) expression in patient tissue samples identified a 2.7-fold reduction in AR expression (p < 0.005) in papillary thyroid cancer compared with matched, normal tissue. An in vitro cell model was developed by stably transfecting AR into 8505C undifferentiated thyroid cancer cells (resulting in clone 84E7). The addition of DHT to the clone 84E7 resulted in AR translocation into the nucleus and a 70% reduction in proliferation, with a shift in the cell cycle toward G1 arrest. RNASeq analysis revealed significant changes in mRNA levels associated with proliferation, cell cycle, and cell cycle regulation. Furthermore, androgen significantly decreased the levels of the G1-associated cell cycle progression proteins cdc25a CDK6 CDK4 and CDK2 as well as increased the levels of the cell cycle inhibitors, p27 and p21. The data strongly suggest that DHT induces a G1 arrest in androgen-responsive thyroid cancer cells. Together, these data support our hypothesis that AR/androgen may play a protective, antiproliferative role and are consistent with younger men having a lower incidence of thyroid cancer than women.

Gene expression profiling identifies potential molecular markers of papillary thyroid carcinoma.

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 expression in papillary thyroid carcinoma promotes invasion via MMP-9 induction.

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.

Inhibition of uPAR and uPA reduces invasion in papillary thyroid carcinoma cells.

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.