plx-4720 and Carcinoma

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong β-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Topics: Animals; Carcinoma; Carcinoma, Papillary; Cellular Senescence; Chromones; Disease Progression; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Indoles; Lymphocytes, Tumor-Infiltrating; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Mice, Transgenic; Morpholines; Mutation, Missense; Neoplasm Proteins; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction; Sulfonamides; Thyroid Neoplasms; Thyrotropin; Tumor Suppressor Protein p53

Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s).

Topics: Antineoplastic Agents; Butadienes; Carcinoma; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Survival; Humans; Indoles; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Invasiveness; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Thyroid Carcinoma, Anaplastic

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

A substantial proportion of solid tumors carry the BRAF V600E mutation, which causes activation of the MEK/MAPK pathway and is a poor prognostic indicator. Patients with locally advanced human cancers are often treated with external beam radiation therapy. Given the association of Raf overactivation with radioresistance, we hypothesized that, in BRAF V600E-mutated carcinomas, there would be combinatorial activity between radiation and PLX4720, a specific BRAF V600E-inhibitor.. Two BRAF V600E-mutated cancer cell lines and one BRAF-V600E wildtype (WT) cancer cell line were obtained. We performed cell viability assays and clonogenic assays using combinations of radiation and PLX4720. We assessed MEK and MAPK phosphorylation at different PLX4720 concentrations with western blotting, and cell cycle progression was evaluated by flow cytometry.. Our results show combinatorial, additive activity between radiation and PLX4720 in BRAF V600E-mutated cell lines, but not in the BRAF WT line. In BRAF V600E-mutated cells, there was a PLX4720 concentration-dependent decrease in MEK and MAPK phosphorylation. In cells with BRAF V600E mutations, PLX4720 caused cell cycle arrest at G1, and, when combined with radiation, caused a combined G1 and G2 cell cycle arrest; this pattern of cell cycle effects was not seen in the BRAF WT cell line.. These data suggest additive, combinatorial activity between radiation and PLX4720 in cancers carrying BRAF V600E mutations. Our data has potential for translation into the multimodality treatment of BRAF V600E-mutated cancers.

Topics: Antineoplastic Agents; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Combined Modality Therapy; Genotype; Humans; Indoles; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Radiation Tolerance; Radiation, Ionizing; Sulfonamides

The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations.. The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines.. We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control.. Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC(50)) ranging from 78-113 nm for PLX4720 and from 29-97 nm for PLX4032. Doses equal to or higher than 500 nm were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G(1) block and altered expression of genes involved in the control of G(1)-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels.. This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

Topics: Adenosine Triphosphate; Binding, Competitive; Carcinoma; Cell Proliferation; Cytostatic Agents; Drug Evaluation, Preclinical; Extracellular Signal-Regulated MAP Kinases; Humans; Indoles; Mutant Proteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Serum; Sulfonamides; Thyroid Neoplasms; Tumor Cells, Cultured