A Cell Proliferation and Chromosomal Instability Signature in

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.


Introduction
Anaplastic thyroid carcinoma (ATC) is a highly malignant tumor that accounts for 2% to 5% of all thyroid cancers and is usually seen in the sixth to seventh decades of life.ATC ranks among the most lethal solid tumors, with a median survival of 4 to 12 months after diagnosis (1)(2)(3).ATC shares genetic alterations with welldifferentiated papillary (PTC) and follicular thyroid carcinomas, namely, point mutations in RAS and BRAF (1-4) and point mutations or gene amplification of PIK3CA (5,6), suggesting that it may derive from a preexisting differentiated lesion.Whereas welldifferentiated thyroid carcinomas are rarely (<10%) p53-mutated, more than 70% of ATC are associated with p53 mutations (2,4).Besides direct gene mutation, several other mechanisms can obstruct p53 function in thyroid cancer, including the upregulation of negative p53 regulators like HMGA1 (high-mobility group A1) and DNp73, or proteins fostering p53 protein degradation, like HDM2 (7,8).
ATC has a high proliferation rate and marked aneuploidy (9).Gene expression profiles correlating with mitotic rate and chromosomal instability in cancer cells have been recently defined.A gene expression signature represented by 168 cell cycle-regulated genes has been identified in in vitro transformed normal human fibroblasts and termed ''the proliferation cluster'' (10).Increased expression of many of these genes, in particular of a core set of 44 of them (''44-gene proliferation cluster''), is a surrogate measure of tumor cell proliferation and is often associated with poor outcome (11).Moreover, the up-regulation of a set of 70 genes, known as the chromosomal instability 70 (''CIN70'') cluster, partially overlapping with the proliferation signature, predicted aneuploidy in several cancers (12).
Genes coding for two protein kinases, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are included in these signatures (10)(11)(12).TTK (also called PYT or hMps1, human monopolar spindle 1 kinase) is a dual-specificity kinase involved in mitotic checkpoint control; it has been implicated in the apoptosis of p53-negative cells after DNA damage (13).PLK1 belongs to the Polo family of serine/threonine kinases.It is important for many cell cyclerelated events, i.e., CDC2 activation, chromosome segregation, centrosome maturation, bipolar spindle formation, activation of the anaphase-promoting complex (APC), and cytokinesis and is overexpressed in many tumors (14).Adoptive overexpression of PLK1 induces NIH 3T3 cell transformation, whereas PLK1 inhibition leads to mitotic arrest and cell death (14).
Here, we show that ATC is characterized by a gene expression profile that overlaps the proliferation and CIN70 signatures, and that PLK1, which is part of these signatures, is essential for survival of ATC cells.

Materials and Methods
Tissue samples.Frozen thyroid tissue samples from 10 patients affected by PTC, 5 patients with ATC, and 4 normal thyroids (N) were used for the microarray screening (Supplementary Table S1).Independent tissue samples (19 normal thyroids, 39 PTC, and 22 ATC) were used for validation assays.All samples were retrieved from the files of the Pathology Department of the University of Pisa (Italy).The study was approved by the Institutional Ethics Committee.The samples were classified according to the diagnostic criteria required for the identification of PTC and ATC (15).The number of neoplastic cells in five areas of 0.2 mm 2 was calculated with a Nikon laser microdissector.
cDNA microarray screening and data analysis.(NIH, Bethesda, MD).The cDNA clones were printed as described elsewhere (16,17).Sample preparation and microarray hybridizations procedures are reported in detail elsewhere (17).Briefly, total RNA was labeled by direct incorporation of Cy5-dUTP or Cy3-dUTP (Amersham Pharmacia Biotech) in a reverse transcription reaction using anchored oligodeoxythymidylate primer (Genosys) and Superscript II reverse transcriptase (Life Technologies Inc.).Cy3-dUTP-tagged cDNAs were mixed with Cy5-dUTP-tagged common reference and subsequently cohybridized to the microarrays.The reference was composed of a pool of RNA from thyroid cell lines and was used throughout all hybridizations to ensure normalized measures for each gene in each individual sample.Hybridized slides were scanned using an Agilent microarray scanner (Agilent Technologies), and images were processed using a collection of IPLab (Scanalytics, Inc.) extensions developed at the Cancer Genetics Branch, National Human Genome Research Institute.The fluorescence intensities of scanned images were quantified, normalized, and corrected to yield the transcript abundance of a gene as an intensity ratio with respect to that of the signal of the references.Genes were ranked according to the weighted gene analysis.Genes with high weight (w) values create greater separation between groups and denser compaction within the groups, i.e., they have a high power to discriminate between normal thyroid, PTC, and ATC.To test the statistical significance of the discriminative weights, sample labels were randomly permutated as previously described (17).This was repeated 1,000 times to generate a w distribution that would be expected under the assumption of random gene expression, i.e., no difference between the groups.Genes that were deemed to significantly (P < 0.001) discriminate between the three categories were listed.The entire data set has been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) 6 and is accessible through GEO series accession number GSE9115.
RNA extraction and quantitative reverse transcription-PCR.Total RNA was isolated with the RNeasy Kit (Qiagen).About 1 Ag of RNA from each sample was reverse-transcribed with the QuantiTect Reverse Transcription (Qiagen).To design a quantitative (Q) reverse transcription-PCR (RT-PCR) assay, we used the Human ProbeLibray system (Exiqon).Primers pairs and PCR conditions are available upon request.PCR reactions were done in triplicate, and fold changes were calculated with the formula: 2 À(sample 1 DCt À sample 2 DCt) , where DCt is the difference between the amplification fluorescent thresholds of the mRNA of interest and the mRNA of RNA polymerase 2 used as an internal reference.
Protein studies.Immunoblotting was carried out according to standard procedures.Anti-PLK1 monoclonal antibody was from Zymed Laboratories; anti-TTK polyclonal antibody was from Santa Cruz Biotechnology; monoclonal anti-a-tubulin was from Sigma-Aldrich; anti-cleaved (Asp 175 ) caspase-3 p17 and p12 fragments polyclonal (5A1) was from Cell Signaling Technology, Inc.; anti-poly(ADP-ribose) polymerase (anti-PARP) monoclonal antibody, which detects full-length PARP and the large fragment (89 kDa) produced by caspase cleavage, was from BD Biosciences.Secondary antimouse and anti-rabbit antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology.
Reporter gene assay.PCR fragments of the human TTK and PLK1 promoters spanning, respectively, from À524 to +72 and from À148 to +63 relative to the transcription start were cloned into the pGL3 Basic vector (Promega Corporation), carrying the Firefly luciferase reporter gene.
Primer sequences are available upon request.Cells were transfected using the LipofectAMINE reagent according to manufacturer's instructions, with 500 ng of the reporter plasmid DNA, together with (when required) p53 wild-type, p21(CIP/WAF1) (25), or E2F4 (kindly donated by M. Crescenzi, Istituto Superiore di Sanita', Rome) expression vectors.A plasmid expressing the enzyme Renilla luciferase (pRL-null) was used as an internal control.In all cases, the total amount of transfected plasmid DNA was normalized by adding empty vector DNA.Forty-eight hours after transfection, Firefly and Renilla luciferase activities were assayed using the Dual-Luciferase Reporter System (Promega Corporation), and the Lumat LB9507 luminometer (EG Berthold).Each experiment was done in triplicate.
RNA silencing.Small inhibitor duplex RNAs targeting PLK1 and the scrambled control [nonspecific small interfering RNA (siRNA) duplex containing the same nucleotides but in irregular sequence] are described elsewhere (26).Cells were grown under standard conditions.The day before transfection, 5 Â 10 4 cells were plated in 35-mm dishes in DMEM supplemented with 10% fetal bovine serum and without antibiotics.Transfection was done using OligofectAMINE reagent (Invitrogen) with 100 nmol/L (250 nmol/L for PC cells) siRNA.Cells were harvested and counted 24 and 48 h after transfection.
Immunofluorescence.Fixed and permeabilized cells were incubated with anti-a-tubulin antibody (Sigma) for 45 min at 37jC.Washed coverslips were incubated with rhodamine-conjugated secondary antibody (Jackson ImmunoResearch) for 30 min at 37jC.After 15 min propidium iodide (1 Ag/ mL) counterstaining and coverslips mounting, stained cells were observed with a Zeiss LSM 510 META confocal microscope.The ''In situ Cell Death Detection Kit, TMR red'' (Roche) was used to detect terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells.At least 300 cells were counted in triplicate experiments.
Fluorescence-activated cell sorter scan analysis.Cells were harvested and fixed in 70% ethanol for 4 h.After washing with PBS, cells were treated with RNase A (100 units/mL) and stained with propidium iodide (25 Ag/mL; Sigma) for 30 min.Samples were analyzed with a CyAn ADP flow cytometer interfaced with the Summit V4.2 software (DakoCytomation).Data were analyzed with the Modfit software (Verity Software House).
Statistical analysis.The two-tailed unpaired Student's t test (normal distributions and equal variances) was used for statistical analysis.All P values were two sided, and differences were significant when P < 0.05.All statistical analyses were carried out using the GraphPad InStat software program (version 3.06.3).

Results
Proliferation-and chromosomal instability-associated genes are up-regulated in ATC.We used cDNA microarrays to characterize the gene expression profile of ATC compared with PTC and normal thyroid tissue samples (Supplementary Table S1).Genes were ranked according to weight values (16,17), with the highest value indicating the most discriminative power (P < 0.001) between the different tissue groups.A set of 914 genes distinguished ATC from normal tissue: 371 of them were up-regulated, and 543 were down-regulated in ATC (Supplementary Table S2).Most of these genes were typical of ATC, and only 114 of them discriminated normal thyroid from both PTC and ATC (Supplementary Tables S2 and S3).
ATC featured the up-regulation of a set of 54 genes involved in the control of cell cycle progression and chromosome segregation; 7 and 13 of them were part of the 44-gene proliferation cluster (10,11) and the CIN70 cluster (12), respectively, and 9 of both clusters (Table 1).These genes were not significantly up-regulated in PTC (Supplementary Table S3).Additional genes of the two clusters tended toward up-regulation in ATC, but the data were not statistically significant (Supplementary Table S4).Thus, ATC overexpressed genes that coded for cyclins (CCNB2,  1).According to recent reports, ATC overexpressed UBE2C (UBCH10), which encodes an E2 ubiquitin-conjugating enzyme that is required for cell cycle progression (27), and genes of the minichromosome maintenance-deficient (MCM) family, involved in licensing DNA for replication (25).Genes encoding other components of the DNA replication initiation complex, such as CDC6, ORC1, and PCNA, also tended toward up-regulation in ATC (Supplementary Table S4).ATC frequently features genetic alterations in the phosphoinositide-3-kinase (PI3K)/AKT pathway (5, 6); accordingly, the screening revealed the altered expression of some genes of this pathway in ATC samples (Supplementary Table S5).Some cis-regulatory DNA elements, e.g., binding sites for E2F and NF-Y (CCAAT box binding) transcription factors and cell cycledependent element (CDE)/cell cycle gene homology region (CHR) DNA orphan binding sites, were highly represented in the promoters of the 54 genes up-regulated in ATC (refs.10, 28-30; Table 1).The E2F family of transcription factors includes positive (E2F1-3) and negative (E2F4-6) regulators of cell cycle progression (28,29).Activator E2Fs (in particular E2F1) are linked to the activation of genes involved in G 1 -S progression, whereas repressor E2Fs (in particular E2F4) bind gene promoters featuring the NF-Y-CDE/CHR DNA module that often peak at the G 2 or G 2 -M phase (10,(28)(29)(30).Many G 2 -M phase genes (like PLK1, TTK, PRC1, CENPE, CENPF, FOXM1, CCNB2, CDC2, CDCA8, CCNA2, TOP2A, UBE2C, and CDC25C) were up-regulated in ATC.Based on such a promoter architecture, it can be argued that some of the gene expression changes associated to ATC may be influenced by the loss of p53 or pRB family gene function, which is able to influence the function of E2F and NF-Y proteins (10,(30)(31)(32)(33). Accordingly, as many as 31 of the 54 genes up-regulated in ATC samples were previously reported to be under p53 negative control (Table 1; refs.13,32,33).Although the p53 gene status of the examined ATC samples is unknown, the microarray screening (Supplementary Table S2) showed that four out of five ATC samples overexpressed HMGA1 (weight = 4.3), a negative regulator of the p53 function (7).
We selected an independent set of ATC samples for validation experiments.A total of 2 out of 10 of these samples harbored R248E and R280T p53 mutations, respectively (data not shown).Moreover, five of them up-regulated HMGA1 mRNA at a RT-PCR analysis (Supplementary Fig. S1).These samples were examined by triplicate quantitative RT-PCR for the expression of 10 of the genes reported in Table 1.The results of these experiments were in agreement with the microarray screening data.Although the expression level of single genes varied among individual ATC samples, these genes were up-regulated in practically all ATC samples examined versus normal thyroids and PTC (Fig. 1A).The difference between ATC and normal tissue was P < 0.001 with the Tukey-Kramer multiple comparisons procedure and P < 0.0001 with the one-way ANOVA test.We also measured the expression levels of the 10 genes in a panel of ATC cell lines expressing a mutated p53 allele (34) in comparison to normal thyroid cells.All of them invariably upregulated in cancer cells (Table 2).PLK1 and TTK transcriptional promoters are negatively controlled by p53 in ATC cells.We focused on PLK1 and TTK, which encode two protein kinases involved in cell cycle progression (13,14).As shown in Table 2, the ATC cell lines analyzed overexpressed PLK1 and TTK compared with the average expression level of normal thyrocytes (P < 0.0001).NPA cells, which are derived from a poorly differentiated thyroid carcinoma, also up-regulated PLK1 and TTK mRNAs.The up-regulation of PLK1 and TTK occurred also at protein level (Fig. 1B).
We cloned the PLK1 gene transcriptional promoter (À148 to +63; ref. 35) and a putative TTK promoter (À524 to +72; ref. 28) in the pGL3 vector upstream from the Firefly luciferase reporter (Fig. 2A).We transiently transfected the pGL3-PLK1-LUC and pGL3-TTK-LUC constructs (or the empty vector) in triplicate in a continuous line of normal thyrocytes (PC) and in ATC cells and measured luciferase activity.Transcription from the TTK (P < 0.0013) and the PLK1 (P < 0.0019) promoters was strongly up-regulated in ATC with respect to normal cells (Fig. 2B).
To better characterize the effects of PLK1 knockdown, we transfected CAL62 cells with PLK1 siRNA or the scrambled control and analyzed them by immunoblot at different time points.Thirtysix hours post-transfection, two biochemical markers of apoptosis, i.e., cleaved products of caspase-3 and of PARP, were visible in PLK1silenced cells but not in cells transfected with the scrambled control (Fig. 4A).Accordingly, PLK1 depletion, but not control RNAi, strongly increased the rate of inter-nucleosomal DNA fragmentation in a TUNEL assay (Fig. 4B).Forty-eight hours after transfection, the percentage of apoptotic cells with a subgenomic DNA (sub-G 1 ) content at the fluorescence-activated cell sorter (FACS) was higher in PLK1-depleted cells than in control cells (14% versus 4%; data not shown).In addition, the fraction of cells with a 4N DNA complement was higher in PLK1-depleted cells (59% versus 14%) versus control cells, which indicates incomplete cytokinesis (data not shown).
We stained PLK1-silenced and control-treated CAL62 cells with anti-a-tubulin antibody and examined the mitotic spindle by confocal microscopy.Consistent with the FACS results, the number of mitotic (prometaphase-like state) cells was increased in PLK1depleted cells as early as 12 h post-transfection.Forty-eight hours after transfection, 53 F 5% of PLK1-depleted cells versus 3 F 2% of control-treated cells were in the M phase (Fig. 4C).As shown in Fig. 4D, PLK1-depleted cells had monopolar (20% versus 0% in control cells) and disorganized (48% versus 9.8% in control cells) spindles.Moreover, in PLK1-depleted cells, there was an increase of bi-nucleated cells (5.4% versus 1.4% in control cells) and cells with an aberrant nuclear morphology (dumbbell-like chromatin; 12% versus 4.3% in control cells; data not shown).A dumbbell-like structure suggests the inability to separate sister chromatids at the onset of anaphase.This feature is a hallmark of so-called mitotic cell death or mitotic catastrophe, a type of apoptosis cells that are committed to when they are unable to complete cytokinesis.

Discussion
This study shows that ATC is characterized by the overexpression of genes associated with cell proliferation and chromosomal instability.This feature, not being detected in welldifferentiated PTC, is typical of ATC and is consistent with the highly mitogenic and aneuploid ATC phenotype (1,2,9).The promoters of most of the proliferation-related genes up-regulated in ATC contain a typical combination of NF-Y and E2F binding sites and CDE/CHR cis-elements, a feature that suggests an indirect control exerted by p53 and pRB tumor suppressors.Consistently, we could show that TTK and PLK1 promoters are negatively controlled by p53-p21(CIP/WAF1) axis in ATC cells.
The CDE/CHR DNA elements are targets of repressor E2Fs (E2F4 and 5; ref. 30).E2F4 is under the negative control of the pRB-like Figure 1.A, expression levels of ATC-specific genes in human thyroid samples.Quantitative RT-PCR analysis of 10 ATC-specific genes in normal thyroid (N ), PTC, and ATC samples.TTK and PLK1 expression was measured in 20 N, 16 PTC, and 13 ATC samples.The other eight genes were measured in five N, nine PTC, and six ATC samples.For each gene, the expression level in a given tumor sample (T ) was obtained by comparing its fluorescence threshold with the average fluorescence thresholds of normal samples (N ).Values are the average results of three independent determinations.B, PLK1 and TTK protein levels in human thyroid cell lines.Fifty micrograms of protein lysate harvested from primary cultures of normal human thyroid follicular (N ) and ATC cells were examined by immunoblot using anti-PLK1 and anti-TTK antibodies.Equal protein loading was verified with an anti-a-tubulin antibody.Data are representative of three independent experiments.p107 (RBL1) and p130 (RB2) proteins, and thus, also E2F4 and pRBlike proteins might be implicated in the regulation of proliferationassociated genes in ATC.We could indeed show that TTK and PLK1 promoters are negatively controlled by E2F4 in ATC cells.
Finally, gene promoters containing NF-Y/CDE-CHR modules often contain ELK1-binding sites (10,28).ELK1 transcription factors are downstream targets of the mitogen-activated protein kinase pathway, and ATC often features mutation in genes (RAS or   BRAF) in this cascade (1)(2)(3)(4).Therefore, it is likely that pathways other than the p53 and pRB ones concurrently regulate the expression of genes of the proliferation cluster.ATC surgery is often only palliative, and there is no effective systemic treatment (2).PLK1 kinase inhibitors are currently being evaluated in clinical trials for various cancer types (14,26,36,37).
Here, we show that PLK1 is required for ATC cell proliferation and survival.This requirement was restricted to ATC cells and was not detected in normal thyroid follicular cells.Although the exact mechanism of this selectivity is unknown, it has been shown that p53 depletion increases the sensitivity of various cell types to PLK1 knockdown (36,37).Whatever the mechanism, our findings indicate that PLK1 addiction might be an Achilles' heel of ATC cells and might be exploited to develop novel treatment strategies for this cancer.
are the average results of three independent determinations.Abbreviation: ND, not done.*Quantitative RT-PCR analysis done as indicated in the legend to Fig.1A.For each gene, the expression level in a given ATC cell line was obtained by comparing its fluorescence threshold with the average fluorescence thresholds of two normal thyroid cell populations (P53N and S11N).cRef.22. bRef.34.x Ref.24.

Figure 2 .
Figure 2. PLK1 and TTK gene promoter activity in ATC cells.A, the two promoters are schematically represented.B, the indicated cell lines were transiently transfected with pGL3-TTK-LUC (left ) or pGL3-PLK1-LUC (right ) plasmids.The average levels of luciferase activity in three independent experiments are reported.Bars, 95% confidence intervals.C, FRO and CAL62 cells were co-transfected with p53, p21(CIP/WAF1), or E2F4 expression vectors (or the empty vector), together with pGL3-TTK-LUC (left) or pGL3-PLK1-LUC (right ).Relative luciferase activity is reported.The average results of three independent assays are reported, and 95% confidence intervals are shown; promoter activity values in vector-transfected cells were arbitrarily set at 100.

Figure 3 .
Figure 3. Effects of PLK1 knockdown on ATC cell growth.CAL62 (A), ARO (B), and normal PC (C ) cells were transfected with either PLK1-siRNA or the scrambled oligonucleotide control.Cells were harvested and counted at the indicated time points.Values represent the average of triplicate determinations.Bars, 95% confidence intervals (right ).Equal amounts of protein lysates were subjected to immunoblot with anti-PLK1 or a-tubulin antibodies (left ).

Figure 4 .
Figure 4. Effects of PLK1 knockdown on apoptosis and cell cycle progression in ATC cells.A, CAL62 cells transfected with either PLK1 siRNA or its scrambled control were lysed at the indicated time points and subjected to immunoblot with anti-cleaved caspase-3 antibodies, which detect only the p17 and p12 cleaved caspase-3 fragments (*) and anti-PARP antibodies, which detect both the intact 116 kDa and its cleaved 89-kDa (*) fragment.Anti-PLK1 immunoblot was used to verify RNAi; a-tubulin levels are shown for normalization.Data are representative of at least three different experiments.B, TUNEL analysis of PLK1-siRNA and scrambled control-treated CAL62 cells (48 h posttransfection).The percentage of positive cells represents the average value of triplicate experiments F SD, in which at least 300 cells were counted.C, CAL62 cells, grown on glass coverslips, were transfected with PLK1-siRNA or with its scrambled control sequence.At the indicated time points, cells were fixed, stained with anti-a-tubulin, and analyzed by confocal microscopy.The percentage of mitotic cells represents the average F SD of three experiments in which at least 400 cells were counted.D, mitotic cells with normal spindles, monopolar spindles, or grossly disorganized spindles were counted.Values refer to the average F SD of triplicate experiments in which at least 200 mitotic cells were counted; insets, representative images.

Table 2 .
Expression levels of ATC-specific genes in human ATC cell lines Expression level (fold change ATC cells versus normal thyrocytes)*