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Supplementary Table 3 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Figure 1 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Table 4 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Table 5 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Methods from Identification of Polo-like Kinase 1 as a Potential Therapeutic Target in Anaplastic Thyroid Carcinoma

Supplementary Table 1 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Table 2 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Figure 1 from Identification of Polo-like Kinase 1 as a Potential Therapeutic Target in Anaplastic Thyroid Carcinoma

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle–dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment. [Cancer Res 2007;67(21):10148–57]

PMID:22919068.-- et al., Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer., This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Ministero dell'Università e della Ricerca (MiUR), and by the grant MERIT of MIUR. G Garcia-Rostan is supported by Programa Ramón y Cajal, Ministerio de Ciencia e Innovación, Social EU Funds, Universidad de Valladolid, Spain. C Nucera (Principal Investigator, Program: Human Thyroid Cancers Preclinical and Translational Research) was funded by the NIHR21CA165039-01A1 and the American Thyroid Association for Thyroid Cancer Research.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and chemoresistant cancers. The serine/threonine kinase Polo-like kinase 1 (PLK1), a key regulator of multiple steps during mitotic progression, is highly expressed in ATC. Here, we used the BI 2536 PLK1 inhibitor on ATC and nontransformed thyroid follicular cell lines. Our data show that ATC cells are addicted to high levels of PLK1 activity for proliferation, survival, anchorage-independent growth, and tumorigenicity. On treatment with nanomolar doses of BI 2536, ATC cells progressed normally through S phase but died thereafter, directly from mitotic arrest. Immunofluorescence microscopy, immunoblot, and flow cytometry analysis showed that, on PLK1 blockade, ATC cells arrested in prometaphase with a 4N DNA content. Treated ATC cells accumulated phosphohistone H3 and displayed characteristic mitotic (Polo) spindle aberrations. Nontransformed thyroid cells were 3.2- to 18.4-fold less susceptible to BI 2536–induced cell cycle effects compared with ATC cells. These findings identify PLK1 as a promising target for the molecular therapy of ATC. [Cancer Res 2009;69(5):1916–23]

Context: The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations. Objective: The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines. Experimental Design: 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. Results: Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC50) 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 G1 block and altered expression of genes involved in the control of G1-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. Conclusions: This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

Purpose: Oncogenic conversion of BRAF occurs in ∼44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. Experimental Design: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six V600EBRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided. Results: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 μmol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho–mitogen-activated protein kinase levels. Conclusions: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the V600EBRAF mutation and, therefore, BRAF suppression might have therapeutic potential in V600EBRAF-positive thyroid cancer.

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.