u-0126 and Carcinoma--Papillary

Long non-coding RNAs (lncRNAs) participate in cancer cell tumorigenesis, cell cycle control, migration, proliferation, apoptosis, metastasis and drug resistance. The BRAF-activated non-coding RNA (BANCR) functions as both an oncogene and a tumor suppressor. Here, we investigated BANCR's role in papillary thyroid carcinoma (PTC) by assessing BANCR levels in PTC and matched normal thyroid epithelial tissues from 92 patients using qRT-PCR. We also used lentiviral vectors to establish PTC cell lines to investigate the effects of BANCR overexpression on cancer cell proliferation, apoptosis, migration and invasion. Our results indicate BANCR levels are lower in PTC tumor tissues than control tissues. Decreased BANCR levels correlate with tumor size, the presence of multifocal lesions and advanced PTC stage. BANCR overexpression reduced PTC cell proliferation and promoted apoptosis, which inhibited metastasis. It also inactivated ERK1/2 and p38, and this effect was enhanced by treatment with the MEK inhibitor U0126. Finally, BANCR overexpression dramatically inhibited tumor growth from PTC cells in xenograft mouse models. These results suggest BANCR inhibits tumorigenesis in PTC and that BANCR levels may be used as a novel prognostic marker.

Topics: Adult; Animals; Apoptosis; Biomarkers, Tumor; Butadienes; Carcinogenesis; Carcinoma, Papillary; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Male; MAP Kinase Signaling System; Mice; Mice, Nude; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Neoplasm Staging; Nitriles; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins B-raf; RNA, Long Noncoding; Thyroid Cancer, Papillary; Thyroid Gland; Thyroid Neoplasms; Xenograft Model Antitumor Assays

The aim of the present study is to investigate the relationship between urinary-type plasminogen activator (uPA) expression and clinicopathological features in papillary thyroid carcinoma (PTC) and to determine the signal transduction of PTC cells in vitro. PTC tissues from 42 patients were analyzed for the expression of uPA and the BRAF(V600E) mutation. BCPAP, a PTC cell line harboring the BRAF(V600E) mutation, was used to study MAPK signaling. PCR and direct sequencing were applied to analyze BRAF(V600E) mutation status. uPA mRNA expression was measured using a quantitative RT-PCR method, and uPA protein was localized using an immunohistochemical method. The ERK protein status was detected by Western blot analysis. uPA gene expression was significantly increased in PTC tissues as compared to the corresponding non-tumor tissues. Furthermore, the up-regulation of uPA mRNAs was correlated with high-risk clinicopathological features, including extrathyroid invasion, loss of cellular polarity/cohesiveness, and the BRAF(V600E) mutation. Marked dephosphorylation of ERK1/2 and down-regulation of uPA expression were detected when BCPAP was treated with a MEK inhibitor, U0126. MEK inhibitors might be a potential treatment strategy for aggressive PTC with BRAF(V600E) through inhibition of uPA expression.

Topics: Adult; Aged; Butadienes; Carcinoma; Carcinoma, Papillary; Down-Regulation; Enzyme Inhibitors; Female; Humans; Male; MAP Kinase Signaling System; Middle Aged; Mutation; Nitriles; Phosphorylation; Proto-Oncogene Proteins B-raf; Thyroid Cancer, Papillary; Thyroid Neoplasms; Up-Regulation; Urokinase-Type Plasminogen Activator

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.

Topics: Antigens, CD; Butadienes; Cadherins; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Movement; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Indoles; MAP Kinase Kinase Kinases; Mutation, Missense; Neoplasm Invasiveness; Nitriles; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Snail Family Transcription Factors; Sulfonamides; Thyroid Cancer, Papillary; Thyroid Neoplasms; Transcription Factors

Thyroid hormone regulates a wide range of cellular activities, including the balance between cell proliferation and differentiation. The thyroid-hormone-inactivating type 3 deiodinase (DIO3, D3) has been shown to be reactivated in human neoplasias. Here, we evaluated DIO3 expression in human papillary thyroid carcinoma (PTC).. Tumor and surrounding normal thyroid tissue were collected from 26 unselected patients with PTC. Clinical data were retrospectively reviewed in medical records. DIO3 mRNA levels were measured by real-time polymerase chain reaction and D3 activity by paper-descendent chromatography. Studies of DIO3 gene regulation were performed in a human PTC-derived cell line (K1 cells). BRAF(V600E) mutation was identified in DNA from paraffin-embedded tissues by direct sequencing. Immunohistochemistry analyses were performed using a specific human D3 antibody.. Increased D3 activity was detected in all 26 PTC samples analyzed as compared with adjacent thyroid tissue. The augmentations in D3 activity were paralleled by increased DIO3 mRNA levels (approximately fivefold). In PTC-derived cells, DIO3 transcripts were further upregulated by the transforming growth factor β1 (TGFβ1). Interestingly, preincubation with mitogen-activated protein kinase (MAPK) cascade inhibitors U0126 (ERK pathway) and SB203580 (p38 pathway) decreased DIO3 mRNA levels and blocked the TGFβ1-induced increase in DIO3 transcripts, suggesting that D3 induction might be mediated through the MAPK signaling pathway. Accordingly, DIO3 mRNA and activity levels were significantly higher in BRAF(V600E)-mutated samples (p=0.001). Increased D3 activity was correlated with tumor size (r=0.68, p=0.003), and associated with lymph node (p=0.03) or distant metastasis (p=0.006) at diagnosis. Conversely, decreased levels of the thyroid-hormone-activating type 2 deiodinase (DIO2) gene were observed in PTC, which might contribute to further decreases in intracellular thyroid hormone levels. Increased D3 expression was also observed in follicular thyroid carcinoma but not in medullary or anaplastic thyroid carcinoma samples.. These results indicate that the malignant transformation of thyroid follicular cell toward PTC promotes opposite changes in DIO3 and DIO2 expression by pretranscriptional mechanisms. The association between increased levels of D3 activity and advanced disease further supports a role for intracellular triiodothyronine concentration on the thyroid tumor cell proliferation or/and dedifferentiation.

Topics: Adult; Butadienes; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Child; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunohistochemistry; Iodide Peroxidase; Male; MAP Kinase Signaling System; Middle Aged; Mutation; Nitriles; Proto-Oncogene Proteins B-raf; Pyridines; Retrospective Studies; Thyroid Cancer, Papillary; Thyroid Neoplasms; Transforming Growth Factor beta1; Young Adult

The V600E BRAF mutant plays an important role in the pathogenesis of papillary thyroid cancer (PTC) and is associated with loss of expression of thyroid iodide-metabolizing genes. This study was done to investigate the restorability of expression of these genes by suppressing the BRAF/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway in V600E BRAF-harboring thyroid cells and to explore the mechanisms involved.. We used inducible expression of V600E BRAF, small interfering RNA transfection, and MEK-specific inhibitor to alter the MAP kinase pathway activities and subsequently examined the changes in expression, promoter activities, and methylation status of thyroid genes.. MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression of V600E BRAF. U0126 also restored the expression of these genes in V600E BRAF-harboring PTC-derived NPA cells. Knockdown of BRAF by specific small interfering RNA restored expression of some of these genes in NPA cells. Luciferase reporter assay using thyroid-stimulating hormone receptor gene as a model showed that the promoter activity was modulated by the MAP kinase pathway. Promoter methylation in association with DNA methyltransferase expression played a role in gene silencing by MAP kinase pathway in NPA cells.. We showed the restorability of expression of thyroid iodide-metabolizing genes silenced by V600E BRAF, and linked this process to gene methylation in PTC cells. The results provide clinical implications that therapeutic targeting at the BRAF/MEK/MAP kinase pathway may be a good approach in restoring thyroid gene expression for effective radioiodine therapy for BRAF mutation-harboring PTC.

Topics: Animals; Butadienes; Carcinoma, Papillary; Gene Expression Regulation, Neoplastic; Humans; Iodides; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; Rats; Receptors, Thyrotropin; RNA, Small Interfering; Thyroid Neoplasms; Transfection

To examine the effects of 2 mitogen-activated protein kinase kinase (MEK1/2) inhibitors on papillary thyroid carcinoma (PTC) cell lines carrying the RET/PTC1 rearrangement or a BRAF mutation. In PTC, RET/PTC1 rearrangement or BRAF mutations results in constitutional activation of RET kinase or BRAF, respectively. Along the RET or BRAF signaling cascades, the activated RET kinase or BRAF activates MEK1/2, and then mitogen-activated protein kinases (extracellular signal-related kinase 1/2 [ERK1/2]) is activated. Activated ERK1/2 enters the nucleus and phosphorylates a variety of transcription factors, resulting in cancer cell proliferation. The MEK1/2 inhibitors, PD98059 and U0126, have been shown to inhibit cell growth in other cancers.. In vitro study.. Papillary thyroid carcinoma cell lines carrying the RET/PTC1 rearrangement (BHP2-7) or a BRAF mutation (BHP5-16).. We treated PTC cells carrying the RET/PTC1 rearrangement or a BRAF mutation with 2 MEK1/2 inhibitors (PD98059 and U0126).. Using Western blot analysis, we detected the expression of phosphorylated ERK1/2 and expression of cleaved poly(ADP-ribose) polymerase (PARP) in cells after treatment with either inhibitors. Growth inhibition was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.. Using Western blot analysis, we detected the dephosphorylation of ERK1/2 in PTC cells carrying the RET/PTC1 rearrangement or a BRAF mutation after treating the cells with 2 MEK1/2 inhibitors (PD98059 and U0126). In addition, both PD98059 and U0126 completely inhibited the growth of the PTC cells carrying a BRAF mutation but partially inhibited the growth of the PTC cells carrying the RET/PTC1 rearrangement. Finally, we observed PARP cleavage only in cells with a BRAF mutation in the Western blot analysis.. These data suggested that treatment with MEK1/2 inhibitors can be used as tools for inhibiting the growth of PTC cells.

Topics: Butadienes; Carcinoma, Papillary; Cell Division; Cell Line, Tumor; Flavonoids; Gene Rearrangement; Humans; Mitogen-Activated Protein Kinase 1; Nitriles; Phosphorylation; Point Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-ret; Thyroid Neoplasms