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 cycledependent 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.

Purpose: Oncogenic conversion of BRAF occurs in f44% 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 ( V600E BRAF) 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 V600E BRAF-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 (IC 50 = 0.5-1 Amol/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 V600E

Here we show that replicative senescence in normal human diploid IMR90 fibroblasts is accompanied by altered expression of a set of microRNAs (miRNAs) (senescence-associated miRNAs), with 14 and 10 miRNAs being either up or downregulated (42-fold), respectively, in senescent with respect to young cells. The expression of most of these miRNAs was also deregulated upon senescence induced by DNA damage (etoposide) or mild oxidative stress (diethylmaleate). Four downregulated miRNAs were part of miRNA family-17, recently associated to human cell and tissue aging. Moreover, eight upregulated and six downregulated miRNAs mapped in specific chromosomal clusters, suggesting common transcriptional regulation. Upon adoptive overexpression, seven upregulated miRNAs induced the formation of senescence-associated heterochromatin foci and senescence-associated b-galactosidase staining (Po0.05), which was accompanied, in the case of five of them, by reduced cell proliferation. Finally, miR-210, miR-376a*, miR-486-5p, miR-494, and miR-542-5p induced double-strand DNA breaks and reactive oxygen species accumulation in transfected cells. In conclusion, we have identified a set of human miRNAs induced during replicative and chemically induced senescence that are able to foster the senescent phenotype by prompting DNA damage. Replicative or cellular senescence, a state of irreversible arrest of cell division, was first described in cultures of human fibroblasts. 1 Since then, replicative senescence has been described in various mammalian cells. 2 The mechanisms underlying senescence include telomere shortening, upregulation of the CDKN1A (p21WAF1) and CDKN2A (p16INK4a and p14ARF) loci, and accumulation of DNA damage. 3 Telomeres become progressively shorter at every round of cell division and this leads to critically short telomere length sensed as double-strand DNA breaks. 4 DNA damage and DNA-damage response (DDR) could be common events to cellular senescence programs initiated by telomere dysfunction and aberrant oncogene activation. 5 Senescent cells are marked by lack of DNA replication; expression of senescence-associated b-galactosidase (SA-b-gal); accumulation of discrete nuclear foci that are termed senescence-associated heterochromatin foci (SAHFs); and senescence-associated DNA-damage foci (SDFs). SAHFs are detected by preferential binding of DNA dyes, such as 4 0 ,6-diamidino-2-phenylindole (DAPI), and the presence of certain heterochromatin-associated histone modifications (trimethyl-Lys9 Histone H3). SDFs are nuclear foci containing proteins that are associated to DNA damage (Ser139-phosphorylated histone H2AX -g-H2AX-and p53-binding protein-1-53BP1). 6 Senescent cells show striking changes in gene expression, including upregulation of cell-cycle inhibitors (p21WAF1 and p16INK4a) and secreted proteins involved in microenvironment remodeling (IL-6), 7 and downregulation of genes that facilitate cell-cycle progression (c-FOS, cyclin-A, cyclin-B, PCNA) 8 or that are involved in cell-cycle execution (FOXM1, UBE2C, TYMS). ...

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]

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 B40% 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.