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Context: Cell lines are a widely used tool in cancer research. However, despite the relatively high incidence of papillary thyroid carcinoma (PTC), there are only four PTC cell lines available for international research audience., Objective: The objective of this study was to establish and characterize new PTC cell lines that represent primary tumor biology. Surgical specimens were obtained to generate PTC cell lines. Short tandem repeat profiling was used to confirm the uniqueness of the cell lines against databases of known cell lines and mutations were assessed using Sequenom. The expression of thyroid-specific genes was examined using real-time PCR. Tumorigenicity was determined using an orthotopic thyroid xenograft tumor mouse model., Results: Six PTC cell lines (five conventional PTCs and one follicular variant of PTC) were generated and found to be unique when compared by short tandem repeat profiling against databases of all existing cell lines. The five conventional PTC cell lines carry the BRAF V600E mutation and the follicular variant of PTC cell line had an NRAS mutation. Five of the six cell lines had a mutation in the promoter of the human telomerase reverse transcriptase gene. None of the cell lines have RET/PTC rearrangements. Three cell lines were tumorigenic in the orthotopic thyroid xenograft tumor mouse model., Conclusions: These five characterized conventional PTC cell lines and the unique follicular variant of PTC cell line should be valuable reagents for thyroid cancer research. The three tumorigenic cell lines can be used for in vivo testing of targeted therapeutic and novel agents.

Context: Special populations of cells that can efficiently initiate tumor growth have been characterized, and this feature supports the cancer stem cell theory. These cancer stem cell populations have been identified with CD44 and POU5F1. Most cancer stem cells express high levels of CD44 and low levels of CD24. In thyroid lesions, cancer stem cells have been detected in anaplastic carcinoma. However, little is known about the presence of cancer stem cells in papillary thyroid carcinoma (PTC), especially in recurrent PTC., Objective and Design: PTC cells were labeled and sorted by flow cytometry to obtain two populations. Total RNA was prepared from cells with high CD44 and CD24 expressions (CD44+CD24+) and from cells with high CD44 and low CD24 expressions (CD44+CD24-). The expressions of the stem cell marker POU5F1 and several differentiated thyroid markers were measured via real-time PCR., Results: CD44+CD24- cells were present in all PTCs tested, and the percentage of these cells was higher in clinically aggressive recurrent PTC than in less aggressive primary PTCs. Higher expression of POU5F1 was found in CD44+CD24- cells compared with that of CD44+CD24+ cells. The expression of POU5F1 was higher in thyrospheroids grown in serum-free condition than in cells grown in the presence of serum from the same patient, and the tumor was initiated in mice using thyrospheroids., Conclusions: The percentage of CD44+CD24- cells varied from tumor to tumor. Our findings suggest that cancer stem cells are present in PTC.

Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy. Most PTC carry one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling transduction pathway leading to cellular proliferation, differentiation, and apoptosis. PD0325901 is a specific MEK1/2 inhibitor and therefore is a promising drug to treat thyroid cancers with either RET/PTC or BRAF mutation. In this study we tested the effects of PD0325901 on PTC cells harboring either mutation in vitro by growth curves and Western blots and in vivo using a murine orthotopic xenograft model. We found that 50% growth inhibition (GI(50)) by PD0325901 was 11 nmol/L for the PTC cells with the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells with a BRAF mutation, with both concentrations readily achievable in serum. After 1 week of oral administration of PD0325901 (20-25 mg/kg/day) in mice, no tumor growth was detected in mice inoculated with PTC cells bearing a BRAF mutation. For PTC with the RET/PTC1 rearrangement, the average tumor volume of the orthotopic tumor was reduced by 58% as compared with controls. In conclusion, our data suggested that PTC cells carrying a BRAF mutation were more sensitive to PD0325901 than were PTC cells carrying the RET/PTC1 rearrangement. Our findings support the clinical evaluation of PD0325901 for patients with PTC and potentially other carcinomas with BRAF mutations., ((c)2010 AACR.)

Background: Papillary thyroid carcinoma (PTC), the most common type of thyroid malignancy, usually possesses mutations, either RET/PTC rearrangement or BRAF mutation. Both mutations can activate the mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling transduction pathway, which results in activation of transcription factors that regulate cellular proliferation, differentiation, and apoptosis., Objective: To test the effects of CI-1040 (PD184352), a specific MEK1/2 inhibitor, on PTC cells carrying either an RET/PTC1 rearrangement or a BRAF mutation., Design: The effects of CI-1040 on PTC cells were evaluated in vitro and in vivo., Main Outcome Measures: The effects of CI-1040 on PTC cells were evaluated in vitro using a cell proliferation assay, cell cycle analysis, and immunoblotting. The antitumor effects of CI-1040 in vivo were evaluated in an orthotopic mouse model., Results: The concentrations of CI-1040 needed to inhibit 50% cell growth were 0.052microM for PTC cells with a BRAF mutation and 1.1microM for PTC cells with the RET/PTC1 rearrangement. After 3 weeks of oral administration of CI-1040 (300 mg/kg/d) to mice with orthotopic tumor implants of PTC cells, the mean tumor volume of implants bearing the RET/PTC1 rearrangement (n = 5) was reduced 47.5% compared with untreated mice (from 701.9 to 368.5 mm(3)), and the mean volume of implants with a BRAF mutation (n = 8) was reduced 31.3% (from 297.3 to 204.2 mm(3))., Conclusions: CI-1040 inhibits PTC cell growth in vitro and in vivo. Because RET/PTC rearrangements are unique to thyroid carcinomas and a high percentage of PTCs possess either mutation, these findings support the clinical evaluation of CI-1040 for patients with PTC.

Purpose: Papillary thyroid carcinoma (PTC), the most common thyroid malignancy, usually possesses BRAF mutation or rearranged in translation (RET)/PTC rearrangements. PTC usually possesses BRAF mutation or RET/PTC rearrangements. The mutation status of patients with recurrent PTC has never been characterized in a large population., Experimental Design: Mutation status was determined in a cohort of 54 patients with recurrent PTC and analyzed for clinicopathologic relationships. BRAF and ras mutations were determined by PCR and sequencing of genomic DNA. RET/PTC rearrangements were analyzed by reverse transcription-PCR., Results: BRAF mutation in exon 15 (V600E) was found in 42/54 (77.8%) recurrent PTC patients. The RET/PTC rearrangements were detected in 9 of 54 (16.7%) patients. In addition, 5 of 54 (9.3%) recurrent PTC patients had both a BRAF mutation and a RET/PTC rearrangement. The prevalence of tumors with dual mutations found in the recurrent population far exceeds the frequency historically reported for patients with primary PTC. Patients with dual mutations were significantly older (80% older than 45 years) than patients with a BRAF mutation alone (38% older than 45 years)., Conclusions: Recurrent PTC is significantly associated with a predominant BRAF mutation. RET/PTC rearrangements, although commonly associated with primary PTCs in younger patients, are uncommonly found in recurrent PTC patients. In addition, the incidence of dual mutations was higher in patients with recurrent PTC than in those primary PTC, as reported by others.

Purpose: Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy with one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the MEK/ERK signaling transduction pathway and result in the activation of transcription factors that regulate cellular proliferation, differentiation, and apoptosis. Sorafenib (Nexavar, BAY 43-9006) is a multikinase inhibitor, and in this study, we tested its effects on PTC cells carrying either mutation., Experimental Design: The effects of sorafenib on cell proliferation and signaling were evaluated in vitro on PTC cells using growth curves, cell cycle analysis, and immunoblotting. Using an orthotopic mouse model, we determined the antitumor effects of sorafenib in vivo., Results: The concentration needed for 50% growth inhibition (GI(50)) by sorafenib was 0.14 mumol/L for the PTC cells with the RET/PTC1 rearrangement, and 2.5 mumol/L for PTC cells with a BRAF mutation, both readily achievable serum concentrations. After 3 weeks of oral administration of sorafenib (80 mg/kg/d) in mice, small (94% reduction compared with controls) or no tumor growth was detected in mice inoculated with PTC cells bearing the RET/PTC1 rearrangement, whereas the tumor volume of the orthotopic tumor implants of PTC cells with a BRAF mutation was reduced 53% to 54% (as compared with controls)., Conclusions: PTC cells carrying the RET/PTC1 rearrangement were more sensitive to sorafenib than PTC cells carrying a BRAF mutation. Because RET/PTC rearrangements are unique to thyroid carcinomas, our findings support the clinical evaluation of sorafenib for patients with PTC and the identification of patients most likely to respond to sorafenib treatment.

Objectives: 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., Design: In vitro study., Subjects: Papillary thyroid carcinoma cell lines carrying the RET/PTC1 rearrangement (BHP2-7) or a BRAF mutation (BHP5-16)., Intervention: We treated PTC cells carrying the RET/PTC1 rearrangement or a BRAF mutation with 2 MEK1/2 inhibitors (PD98059 and U0126)., Main Outcome Measures: 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., Results: 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., Conclusion: These data suggested that treatment with MEK1/2 inhibitors can be used as tools for inhibiting the growth of PTC cells.