Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines.

Anaplastic thyroid cancers (ATC) are aggressive tumors, which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy, and most patients die within a few months of diagnosis. In the present study, we evaluated the in vitro effects on ATC cells of VX-680, an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy were tested on the ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time- and dose-dependent manner, with the IC50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with > or =4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion, our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment.

Transforming acidic coiled-coil 3 and Aurora-A interact in human thyrocytes and their expression is deregulated in thyroid cancer tissues.

Aurora-A kinase has recently been shown to be deregulated in thyroid cancer cells and tissues. Among the Aurora-A substrates identified, transforming acidic coiled-coil (TACC3), a member of the TACC family, plays an important role in cell cycle progression and alterations of its expression occur in different cancer tissues. In this study, we demonstrated the expression of the TACC3 gene in normal human thyroid cells (HTU5), and its modulation at both mRNA and protein levels during cell cycle. Its expression was found, with respect to HTU5 cells, unchanged in cells derived from a benign thyroid follicular tumor (HTU42), and significantly reduced in cell lines derived from follicular (FTC-133), papillary (B-CPAP), and anaplastic thyroid carcinomas (CAL-62 and 8305C). Moreover, in 16 differentiated thyroid cancer tissues, TACC3 mRNA levels were found, with respect to normal matched tissues, reduced by twofold in 56% of cases and increased by twofold in 44% of cases. In the same tissues, a correlation between the expression of the TACC3 and Aurora-A mRNAs was observed. TACC3 and Aurora-A interact in vivo in thyroid cells and both proteins localized onto the mitotic structure of thyroid cells. Finally, TACC3 localization on spindle microtubule was no more observed following the inhibition of Aurora kinase activity by VX-680. We propose that Aurora-A and TACC3 interaction is important to control the mitotic spindle organization required for proper chromosome segregation.

Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors.

Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

Expression of Aurora kinases in human thyroid carcinoma cell lines and tissues.

The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. In the present study, we demonstrated that human thyrocytes express all 3 Aurora kinases (A, B and C) at both protein and mRNA level and this expression is cell cycle-regulated. An increase in the protein level of the 3 kinases was found, with respect to normal human thyrocytes (HTU5), in the human cell lines derived from follicular (FTC-133), papillary (B-CPAP) and anaplastic (8305C) thyroid carcinomas, but not in cells derived from a follicular adenoma (HTU42). These observations were mirrored in RT-PCR experiments for Aurora-A and B. In contrast, Aurora-C mRNA levels were not significantly different among the different cell types analyzed, suggesting that posttranscriptional mechanism(s) modulate its expression. The expression at the protein level of all 3 Aurora kinases was significantly higher in 3 thyroid papillary carcinomas with respect to normal matched tissues obtained from the same patients. Similar modifications, at the mRNA level, could be observed in 7 papillary carcinoma tissues for Aurora-A and B, but not for Aurora-C. In conclusion, we demonstrated that normal human thyrocytes express all 3 members of the Aurora kinase family, and their expression is amplified in malignant thyroid cell lines and tissues. These results suggest that the Aurora kinases may play a relevant role in malignant thyroid cancers, and may represent a putative therapeutic target for thyroid neoplasms.

HPSE-1 expression and functionality in differentiating neural cells.

The study of cellular differentiation encompasses many vital parts of biology and medicine. Heparan sulfate proteoglycans (HSPG) are essential and ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells and tissues. Heparan sulfate chains (HS) of HSPG bind and sequester a multitude of extracellular ligands, including growth factors, cytokines, chemokines, enzymes, and lipoproteins. Enzymatic degradation of HS is therefore involved in processes such as cell proliferation, migration, and differentiation. Heparanase (HPSE-1) is an HS degradative enzyme associated with inflammation and lipid metabolism and is a critical molecular determinant in cancer metastasis. The enzyme acts as an endo-beta-D-glucuronidase, which degrades HS at specific intrachain sites, resulting in HS fragments of discrete molecular weights that retain biological function. HPSE-1's relevance as the only example of cloned/purified mammalian HS degradative enzyme led us to investigate its functionality in human olfactory epithelium (HOE) cells as a paradigm for HPSE-1's roles in neural cell differentiation. We provide the first evidence of 1) HPSE-1 presence in HOE cells and 2) a highly significant increase of HPSE-1 mRNA and enzyme activity in differentiating vs. proliferating HOE cells. Our data suggest that an augmented HPSE-1 activity may represent a physiological mechanism involved in neural cellular differentiation.

During apoptosis of tumor cells HMGA1a protein undergoes methylation: identification of the modification site by mass spectrometry.

Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.