Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid ...carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)β in mediating this process. Accordingly, TGFβ-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFβ-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFβ-induced EMT, through a MAPK-dependent process.
Exposure to ionizing radiation during childhood markedly increases the risk of developing papillary thyroid cancer. We examined tissues from 26 Ukrainian patients with thyroid cancer who were younger ...than 10 years of age and living in contaminated areas during the time of the Chernobyl nuclear reactor accident. We identified nonoverlapping somatic driver mutations in all 26 cases through candidate gene assays and next-generation RNA sequencing. We found that 22 tumors harbored fusion oncogenes that arose primarily through intrachromosomal rearrangements. Altogether, 23 of the oncogenic drivers identified in this cohort aberrantly activate MAPK signaling, including the 2 somatic rearrangements resulting in fusion of transcription factor ETS variant 6 (ETV6) with neurotrophic tyrosine kinase receptor, type 3 (NTRK3) and fusion of acylglycerol kinase (AGK) with BRAF. Two other tumors harbored distinct fusions leading to overexpression of the nuclear receptor PPARγ. Fusion oncogenes were less prevalent in tumors from a cohort of children with pediatric thyroid cancers that had not been exposed to radiation but were from the same geographical regions. Radiation-induced thyroid cancers provide a paradigm of tumorigenesis driven by fusion oncogenes that activate MAPK signaling or, less frequently, a PPARγ-driven transcriptional program.
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•New compositional and isotopic constraints on the origin of chromitites at Finero.•Laurite have similar unradiogenic 187Os/188Os values and TRD ages ca. 450Ma or older.•Trace-element ...signatures of zircons support carbonatite nature of metasomatism.•Zircon U-Pb ages record prolonged mantle metasomatism from ∼310Ma to 190Ma.•Zircon Hf-isotope composition implies derivation from a relatively old source (ca. 0.9Ga).
Chromitites enclosed within metasomatised Finero phlogopite peridotite (FPP) contain accessory platinum-group minerals, base metal (BM) sulfides, baddeleyite, zircon, zirconolite, uraninite and thorianite. To provide new insights into mantle-crustal interaction in the Finero lithosphere this study evaluates (1) the mineral chemistry and Os-isotope composition of laurite, (2) the crystal morphology, internal structure, in-situ U-Pb, trace-element and Hf-isotope data of zircon from two chromitite localities at Alpe Polunia and Rio Creves. The osmium isotope results reveal a resticted range of ‘unradiogenic’ 187Os/188Os values for laurite at Alpe Polunia (0.1247–0.1251, mean 0.1249±0.0001). Re-Os model ages (TRD) of laurite reflect an Early Paleozoic partial melting event (ca 450Ma or older), presumably before the Variscan orogeny. The Os isotopic composition of laurite/chromitite probably preserves their mantle signature and was not affected by later metasomatic processes. U-Pb and Hf-isotope data show that the Finero chromitites have distinct zircon populations with peculiar morphology, internal cathodoluminescence textures, trace-element composition and an overall U-Pb age span from ∼310Ma to 190Ma. Three age peaks at Rio Creves (220±4Ma, 234.2±4.5Ma and 277.5±3.2Ma) are consistent with a prolonged formation and multistage zircon growth, in contrast to the common assumption of a single metasomatic event during chromitite formation. The trace-element signatures of zircons are comparable with those of mantle-derived zircons from alkaline ultramafic rocks, supporting the carbonatitic nature of the metasomatism. Hf-isotope compositions of the Finero zircons, with εHf values ranging mainly from −3 to +1, are consistent with crustal input during metasomatism and could indicate that the parental melts/fluids were derived from a relatively old source; the minimum estimates for Hf model ages are 0.8–1.0Ga. Our findings imply that mantle rocks and metasomatic events at Finero have a far more complex geological history than is commonly assumed.
Information about the clinical efficacy and complications of the circumferential mapping and isolation of the pulmonary veins (PVs) in patients with atrial fibrillation (AF) is still limited. The ...present study included 75 patients (mean age 58 ± 11 years, 20 women) with paroxysmal (n = 69) or persistent AF (n = 6). Mapping of PVs was performed with a circumferential mapping catheter. After preferential PV–left atrium (LA) electric inputs were defined, radiofrequency ablation was performed until complete isolation of the PVs from the LA was achieved. A total of 226 PVs were mapped; 195 (86%) showed typical PV potentials. Complete isolation of PVs from the LA was achieved in 173 PVs (89%). Detailed follow-up, including 7-day Holter monitoring at 1, 4, 9, and 12 months after intervention was performed. If AF reoccurred, PVs were mapped and reisolated. After a mean follow-up period of 230 ± 133 days, 38 of 75 patients (51%) were in sinus rhythm. At 1, 4, and 9 months of follow-up, 31 of 65 patients (48%), 36 of 53 patients (68%, p = 0.04 as compared with the first month), and 21 of 28 patients (75%, p = 0.025 as compared with the first month), respectively, were in sinus rhythm. During follow-up, 30 patients (40%) underwent a second ablation procedure due to recurrence. Recurrences were related to resumption of PV muscle–left atrial conduction (27 patients) and/or extra PV foci (12 patients) or nonablated PVs (8 patients). Complications occurred in 17 patients (22%). PV stenosis was detected in 13 patients (25% to 50% in 7 patients and >50% in 6 patients). Pericardial effusion occurred in 4 patients. It was concluded that isolation of the PV from the LA is moderately effective in the prevention of AF recurrence and could be associated with serious acute and long-term complications.
Inactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify ...a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown.
We screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAP
, alone or in combination with endogenous expression of either Hras
or Braf
. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAP
. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib.
We found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells.
YAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAF
-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-
cancers.
Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- ...contaminated.
Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis.
Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines.
Conclusions: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.
Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and ...detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T→A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.
Abstract
Context
BRAFV600E mutant thyroid cancers are often refractory to radioiodine (RAI).
Objectives
To investigate the utility and molecular underpinnings of enhancing lesional iodide uptake with ...the BRAF inhibitor vemurafenib in patients with RAI-refractory (RAIR).
Design
This was a pilot trial that enrolled from June 2014 to January 2016.
Setting
Academic cancer center.
Patients
Patients with RAIR, BRAF mutant thyroid cancer.
Intervention
Patients underwent thyrotropin-stimulated iodine-124 (124I) positron emission tomography scans before and after ~4 weeks of vemurafenib. Those with increased RAI concentration exceeding a predefined lesional dosimetry threshold (124I responders) were treated with iodine-131 (131I). Response was evaluated with imaging and serum thyroglobulin. Three patients underwent research biopsies to evaluate the impact of vemurafenib on mitogen-activated protein kinase (MAPK) signaling and thyroid differentiation.
Main Outcome Measure
The proportion of patients in whom vemurafenib increased RAI incorporation to warrant 131I.
Results
Twelve BRAF mutant patients were enrolled; 10 were evaluable. Four patients were 124I responders on vemurafenib and treated with 131I, resulting in tumor regressions at 6 months. Analysis of research tumor biopsies demonstrated that vemurafenib inhibition of the MAPK pathway was associated with increased thyroid gene expression and RAI uptake. The mean pretreatment serum thyroglobulin value was higher among 124I responders than among nonresponders (30.6 vs 1.0 ng/mL; P = 0.0048).
Conclusions
Vemurafenib restores RAI uptake and efficacy in a subset of BRAF mutant RAIR patients, probably by upregulating thyroid-specific gene expression via MAPK pathway inhibition. Higher baseline thyroglobulin values among responders suggest that tumor differentiation status may be a predictor of vemurafenib benefit.
The BRAF inhibitor vemurafenib increased radioiodine uptake in a subset of patients with BRAF mutant, radioiodine-refractory thyroid cancer.
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