A novel amplification target, DUSP26, promotes anaplastic thyroid cancer cell growth by inhibiting p38 MAPK activity

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Abstract

Anaplastic thyroid cancer (ATC) is one of the most lethal of all human tumors, but cytogenetic information concerning ATC is extremely limited. Using our in-house array-based comparative genomic hybridization and 14 ATC cell lines with further fluorescence in situ hybridization analysis, we demonstrated amplification of the DUSP26 gene, known by another report as MAP kinase phosphatase-8. DUSP26 was overexpressed in ATC cell lines and primary ATC tumor samples. When overexpressed, either exogenously or endogenously, DUSP26 promoted growth of the ATC cells. DUSP26 encodes a protein containing a dual-specificity phosphatase domain that can dephosphorylate itself. DUSP26 effectively dephosphorylates p38 and has a little effect on extracellular signal-regulated kinase in ATC cells. DUSP26 protein formed a physical complex with p38, and promoted survival of ATC cells by inhibiting p38-mediated apoptosis. Our findings suggest that DUSP26 may act as an oncogene in ATC, and might be a useful diagnostic marker and therapeutic target of this disease.

Introduction

Thyroid cancers are classified as medullary, papillary, follicular or anaplastic. Anaplastic thyroid carcinoma (ATC) is one of the most virulent of all human malignancies (Sherman, 2003), with a mean survival time among patients of less than a year after diagnosis (Passler et al., 1999), but cytogenetic information about it is very limited. As multiple genetic alterations occurring sequentially in a cell lineage underlie the carcinogenetic process in solid tumors, unraveling the molecular mechanisms in this process could provide pivotal diagnostic biomarkers and targets for developing more effective agents for this heretofore quickly fatal disease. However, only a few studies on the molecular aspects of ATC, including analysis of genome-wide gene expression (Onda et al., 2004), have been carried out, because ideal biological material is hard to obtain given that most ATC tumors are not subject to surgical intervention (Wiseman et al., 2003).

Extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK) belong to a molecular family of mitogen-activated protein kinases (MAPKs). These kinases are thought of as functionally distinct. ERK is thought to be involved in cell proliferation (Widmann et al., 1999). On the other hand, a recent study highlighted p38 as a negative regulator of cell-cycle progression along with its previously well-characterized proapoptotic functions (Bulavin and Fornace, 2004a), although it has been shown that p38-MAPK cascades enhance survival (Liu et al., 2001; Park et al., 2002), cell growth (Juretic et al., 2001) and differentiation (Yosimichi et al., 2001). The coordinated balance between ERK and p38 activity may represent a critical mechanism for controlling cell-fate decisions as well (McMullen et al., 2005). Therefore, these findings suggest that the p38-MAPK pathway may be required for apoptosis and survival depending on cell types and conditions (Wada and Penninger, 2004). Inactivation of activated MAPKs is important for normal cellular responses and can be achieved by dephosphorylation of either the threonine or the tyrosine residue, or both. Various dual-specificity protein phosphatases (DSPs) are known to dephosphorylate both residues of MAPKs (Tanoue et al., 2001). By dephosphorylating specific MAPKs, many DSPs can function as oncoproteins or tumor suppressors by modifying the MAPK pathway (Dixon et al., 1998; Hoornaert et al., 2003). Overexpression of some DSPs can protect against apoptosis in cancer cells (Srikanth et al., 1999; Wu et al., 2004).

Amplification of chromosomal DNA is one of the mechanisms capable of activating genes that are implicated in tumor development (Bishop, 1991). As proto-oncogenes contribute to the malignant phenotype in a dominant manner through overexpression of the gene product, or by expression of a protein product with altered function (Weinberg, 1995), identification of amplified regions and the genes affected by them may represent a promising route toward revealing which genes are likely to contribute to the carcinogenesis of ATC. In this study, we analysed genomic copy-number aberrations in 14 ATC cell lines using our in-house array-based comparative genomic hybridization (array-CGH) system (Inazawa et al., 2004; Izumi et al., 2005; Takada et al., 2005), and detected amplification of a gene DUSP26, which had recently been reported by others as MAP kinase phosphatase (MKP-8) (Vasudevan et al., 2005). The gene product contains a dual-specificity phosphatase domain and, when expressed exogenously or endogenously, DUSP26 was shown to promote growth and/or inhibit death of ATC cells. As some DSPs, such as MKP-1, were shown to promote cell survival by attenuating stress-responsive MAPK-mediated apoptosis (Wu and Bennett, 2005), we further analysed the association between DUSP26-mediated MAPK dephosphorylation and cell growth or survival, and demonstrated that DUSP26 promotes survival of ATC cells by inhibiting p38MAPK activity and inhibiting p38-mediated apoptosis. Together, these observations suggest that DUSP26 has an oncogenic function in ATC.

Results

Identification of an 8p12 amplicon in ATC cell lines by array-CGH analysis

Through screening DNA copy-number aberrations in 14 ATC cell lines by array-CGH analysis, six clones/genes showing amplifications (log2 ratio >2) were detected (data not shown): five of them (MET, MYC, PVT1, YAP1 and CIAP1) had been mapped to amplicons reported previously in various human cancers (Walentinsson et al., 2001; Ray et al., 2004; Snijders et al., 2005). To identify novel genes critical to ATC, we focused on the P1-artificial chromosome (PAC) RP1-25D10 clone mapped to 8p12, which was amplified in cell line 8305C (Figure 1a). To define a map of this amplification, we performed fluorescence in situ hybridization (FISH) experiments with 8305C (Figure 1b) and four other ATC cell lines (8505C, KTA1, KTA2 and HTC/C3), using 10 bacterial artificial chromosomes (BACs) mapped near RP1-25D10 as probes. Relative positions of these BACs on a map of the 8p12 region are indicated in Figure 1c. Copy numbers, as well as molecular organization of the amplicon, were assessed by analysis of the hybridization patterns on metaphase and interphase chromosomes.

Figure 1
figure1

Amplification and overexpression of DUSP26 in ATC cell lines. (a) Representative array-CGH array image (top panel) and genetic changes observed on whole chromosomes (bottom panel) of the 8305C cell line. A remarkable increase in copy number of the PAC clone RP1-25D10 at 8p12 was detected as a clear green signal (log2 ratio=3.2, red arrows). (b) Representative FISH image with the BAC RP11-258M15 as a probe hybridized to metaphase chromosomes from 8305C cell line, showing a dmins pattern. (c) Left: map of 8p12 covering the region amplified in ATC cell lines. Ten BACs used for FISH are indicated as vertical bars. Five transcripts present in the amplicon are indicated. All markers and transcripts are positioned according to the genome database (http://genome.ucsc.edu/, May 2004 assembly). Center: summarized results of DNA copy-number analyses by FISH. The horizontal axis (top) indicates the number of FISH signals detected by each BAC probe; the number of signals was truncated at 20 because it was difficult to enumerate them above that level. Right: relative position of four amplicons detected at 8p11–12 in three different cancers. Total bar, 8p11–12 amplicon detected in breast cancer (Ray et al., 2004); ATC bar, 8p12 amplicon detected by this study; breast-cancer bar, 8p12 amplicon detected by Garcia et al. (2005); lung cancer bar, 8p12 amplicon detected by Tonon et al. (2005). (d) RT–PCR to detect levels of mRNA expression of five transcripts present in the 8p12 amplicon among 14 ATC cell lines. Expression levels of DUSP26 and RNF122 correlated well with their copy-number status. The copy numbers were determined by FISH experiments using BAC RP11-107B2 (containing DUSP26 and RNF122) as a probe. (e) Expression of DUSP26 mRNA in seven primary ATCs and normal thyroid tissue, measured by RT–PCR. PCR products were electrophoresed in 3% agarose gel. The band quantification was performed using LAS-3000 (Fujifilm, Tokyo, Japan) and Multi Gauge software (Fujifilm). The expression level of DUSP26, normalized with GAPDH, in each sample was divided by that in normal thyroid, and recorded as a fold increase.

In cell line 8305C, five BACs (RP11-451O18, 722E23, 107B2, 258M15 and 352N23) produced the highest number of signals with a pattern of double minute chromosomes (dmins) as shown in Figure 1b. Fewer signals were detected with the other five BACs in the region (RP11-692J15, 11N9, 248O9, 155H15 and 79H13), suggesting that the latter were located outside the amplicon. Four additional cell lines yielded three signals (KTA1) or four signals (KTA2, 8505C and HTC/C3). Therefore, the smallest region of overlap (SRO) could lie between BAC451O18 and BAC352N23 (Figure 1c). The SRO is about 1 Mb in extent, according to the information in the human genome databases (http://www.ncbi.nlm.nih.gov/ and http://genome.ucsc.edu/). Figure 1c shows that the SRO of ATC is different from other 8p11–12 amplicons that have been detected in breast and lung cancers (Ray et al., 2004; Garcia et al., 2005; Tonon et al., 2005).

Identification and cloning of genes affected by the amplification

Genes activated by genomic copy number increases and involved in the progression of tumors are likely to be located in the SRO of amplicons. We performed reverse transcriptase–polymerase chain reaction (RT–PCR) with all of the five transcripts located within the SRO (Figure 1c) to determine the expression status of them in 14 ATC cell lines. Among these five transcripts, only DUSP26 and RNF122 were consistently overexpressed in the ATC cell lines that had exhibited increased copy numbers compared with cell lines exhibiting normal copy numbers (Figure 1d), suggesting that the two transcripts might be targets for gain/amplification at 8p12. On the other hand, the other three transcripts (FUT10, LOC84549 and FLJ23263) were consistently expressed regardless of copy-number gains in the region.

For characterization of potential oncogenic properties of DUSP26 and RNF122, we cloned both full-length cDNAs into mammalian and bacterial expression vectors. As epitope-tagged RNF122 protein could not be successfully expressed in either mammalian or bacterial cells (data not shown), we focused on DUSP26 for further characterization. Although ATC is so aggressive that surgical intervention to provide fresh tissue for analysis is rare, we have been able to determine levels of DUSP26 mRNA in seven primary ATC tumors, and DUSP26 was overexpressed (fold increase >2.0) in at least three of them compared with normal thyroid tissue (Figure 1e).

The predicted product, DUSP26, contains a dual-specificity phosphatase domain. During the progression of this study, another group reported the cDNA sequence of the same gene and termed it MKP-8 as a functional phosphatase for MAPK (Vasudevan et al., 2005).

Oncogenic activity of DUSP26

To investigate the effect of DUSP26 on the growth of ATC cells, colony-formation assays were performed using KTA1, KTA3 and TTA1 cells, which exhibit no amplification and low expression of DUSP26 mRNA (Figure 1d). Transiently transfected pCMV-Tag3B-DUSP26 produced markedly more colonies than did the control plasmid (pCMV-Tag3B-mock; Figure 2a).

Figure 2
figure2

Effect of DUSP26 on growth of ATC cells. (a) Colony-formation assay of KTA1, KTA3 and TTA1 cells, which had shown low levels of DUSP26 expression. Left: Two weeks after transfection and subsequent selection of drug-resistant colonies in six-well plates, DUSP26 produced markedly more colonies than did mock. Right: Quantitative analysis of colony formation. Colonies >2 mm were counted, and the results are presented as the mean±s.e.m. of three separate experiments, each performed in triplicate. Statistical analysis used the Mann–Whitney U-test: *DUSP26-transfected cells versus mock-transfected cells. All P<0.05. (b) Suppression of DUSP26 mRNA expression by DUSP26-specific siRNA in 8305C, 8505C and HTC/C3 cells, which natively express the gene at high levels. RT–PCR was performed 48 h after transfection. (c) Effect of DUSP26-specific siRNA on the growth of 8305C, 8505C and HTC/C3 cells measured by WST assay. The results are presented as the mean±s.e.m. of three separate experiments. Statistical analysis used the Mann–Whitney U-test: *DUSP26-specific siRNA-transfected cells versus nonspecific control siRNA-transfected cells. All P<0.05. (d) Effect of DUSP26-specific siRNA on the number of dead 8305C and 8505C cells 72 h after transfection, as determined by direct counting using the trypan blue exclusion method. The results are presented as the mean±s.e.m. of three separate experiments. Statistical analysis used the Mann–Whitney U-test: *DUSP26-specific siRNA-transfected cells versus nonspecific control siRNA-transfected cells. All P<0.05. (e) Typical changes in nuclear morphology observed 72 h after transfection of 8305C cells with DUSP26-specific siRNA or nonspecific control siRNA.

To test whether suppression of endogenously expressed DUSP26 might suppress growth of ATC cells, a DUSP26-specific small interfering RNA (siRNA) was transfected into 8305C, 8505C and HTC/C3 cell lines that express DUSP26 mRNA at high levels (Figure 1d). RT–PCR analysis demonstrated that DUSP26-specific siRNA reduced endogenous DUSP26 mRNA expression compared with nonspecific control siRNA (Figure 2b). In all three cell lines tested, cell growth was remarkably inhibited by DUSP26-specific siRNA compared with nonspecific control siRNA (Figure 2c). Moreover, DUSP26-specific siRNA induced a marked increase in dead cells (Figure 2d); cells with typical apoptotic changes, such as condensation or fragmentation of nuclear chromatin, were observed more frequently in 8305C cultures treated with DUSP26-specific siRNA than in nonspecific control cultures (Figure 2e).

Oncogenic activity of DUSP26 depends on its phosphatase activity

Recombinant 6xHis-tagged wild-type and catalytically inactive mutant of DUSP26 (C151S) were purified from Escherichia coli (Figure 3a) and tested for phosphatase activity using p-nitrophenyl phosphate (pNPP) as a substrate. Consistent with the results in recent reports (Vasudevan et al., 2005; Hu and Mivechi, 2006), wild-type DUSP26 protein could dephosphorylate pNPP, whereas the catalytically inactive mutant demonstrated no phosphatase activity (Figure 3b).

Figure 3
figure3

Oncogenic activity of DUSP26 depends on its phosphatase activity. (a) SDS–PAGE gel analysis of 6xHis-DUSP26wt and 6xHis-DUSP26mt proteins purified from E. coli. (b) In vitro phosphatase activity of 6xHis-DUSP26wt and 6xHis-DUSP26mt proteins using pNPP as a substrate, and the results are presented as the mean±s.e.m. of three separate experiments. (c) Western blotting analysis of DUSP26wt and DUSP26mt expression in stably transfected clones of the KTA3 cells. Note that a retarded gel mobility of the DUSP26mt protein, compared to DUSP26wt, was observed, suggesting that some modification was added to DUSP26mt protein in vivo. (d) Growth rates of KTA3-DUSP26wt and KTA3-DUSP26mt cells, which stably express Myc-tagged DUSP26wt protein and Myc-tagged DUSP26mt protein, respectively. The results are the mean±s.e.m. of three separate experiments. Statistical analysis used the Mann–Whitney U-test: a, KTA3-DUSP26wt cells versus mock-transfected KTA3 cells; b, KTA3-DUSP26wt cells versus KTA3-DUSP26mt cells. All P<0.05.

For in vivo assay, we established clones stably expressing DUSP26wt, DUSP26mt or empty vector into KTA3 cells (KTA3-DUSP26wt, KTA3-DUSP26mt, KTA3-mock cells, respectively). DUSP26wt and DUSP26mt protein expression was detected in lysates from KTA3-DUSP26wt and KTA3-DUSP26mt clones, respectively. Interestingly, a retarded gel mobility of the DUSP26mt protein, compared to DUSP26wt, was observed (Figure 3c), although this mobility shift had not been observed between DUSP26wt and DUSP26mt recombinant proteins purified from E. coli (Figure 3a), suggesting that some modification was added to DUSP26mt protein in vivo.

To test whether the oncogenic property of DUSP26 depends on its phosphatase activity, we performed a proliferation assay. KTA3-DUSP26wt cells showed an increased growth rate compared with KTA3-DUSP26mt or KTA3-mock cells (Figure 3d).

DUSP26 dephosphorylates p38 effectively in ATC cells

As dual-specificity phosphatases can dephosphorylate and inactivate MAPKs (Tanoue et al., 2001), we assessed the kinetics of ERK, p38 and JNK activities in ATC cells. KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells were starved of serum for 24 h, and then stimulated with 10% fetal bovine serum (FBS) for 30 min. Following stimulation, JNK was activated in KTA3-DUSP26mt and KTA3-mock cells to levels equivalent to that of KTA3-DUSP26wt cells (Figure 4a). In contrast, p38 and ERK were hyper-activated in KTA3-DUSP26mt and KTA3-mock cells compared with KTA3-DUSP26wt cells, but the effect of DUSP26 on p38 was more remarkable than ERK (Figure 4a), suggesting that DUSP26 is a more critical negative regulator for p38 than ERK, but not at all for JNK, in ATC cells stimulated with FBS. Moreover, 8305C and 8505C cells transfected for 72 h with DUSP26-specific siRNA showed a markedly increase in p38 activation and a slight increase in ERK activation, compared with nonspecific control siRNA (Figure 4b). These results were consistent with recently published results showing that DUSP26 had little effect on ERK and JNK, but had a significant effect on p38 kinase activity in 293T cells (Vasudevan et al., 2005), although the conflicting finding showing that DUSP26 specifically inactivated ERK1/2 and JNK1, but not p38, was reported very recently (Hu and Mivechi, 2006).

Figure 4
figure4

DUSP26 dephosphorylates p38 efficiently in ATC cells. (a) KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells were serum-deprived for 24 h (left) and stimulated with 10% FBS 30 min (right). Activation of ERK, p38 and JNK was determined with anti-phospho-ERK, anti-phospho-p38 or anti-phospho-JNK antibodies, respectively, and immunoblots were re-probed with anti-ERK, anti-p38 or anti-JNK antibodies. (b) Effect of DUSP26-specific siRNA on activation of p38, ERK and JNK in 8305C and 8505C cells transfected for 72 h. Lysates were collected and submitted to Western blotting with appropriate antibodies. The band quantification was performed using LAS-3000 (Fujifilm, Tokyo, Japan) and Multi-Gauge software (Fujifilm). Intensity of phosphorylated form of each MAPK was normalized by dividing it by that of the corresponding total MAPK, and relative ratio of DUSP26-specific siRNA cells compared to corresponding nonspecific control siRNA were calculated and recorded. (c) Dephosphorylation of p38 by DUSP26 in vitro. Phosphorylated p38 was purified from anisomycin-treated KTA3 cells and incubated with purified 6xHis-DUSP26wt and 6xHis-DUSP26mt proteins. The phosphorylation state of p38 was examined by immunoblotting with anti-phospho-p38 antibody. (d) Pull-down assay using His and His-DUSP26. KTA3 cells were lysed after treatment with 10 μg/ml of anisomycin for 30 min, and then incubated with His or His-DUSP26 fusion proteins immobilized on Ni–NTA superflow affinity resin. The resins were immunoblotted with p38 antibody.

To confirm whether p38 can serve as a direct substrate for DUSP26, we first performed in vitro dephosphorylatoin assay. KTA3 cells were stimulated with anisomycin and the activated p38 was immunopurified and incubated with 6xHis-DUSP26wt or 6xHis-DUSP26mt in vitro; then, the remaining phosphorylated p38 was measured with an immunoblot analysis using anti-phospho-p38 antibody. As shown in Figure 4c, wild-type DUSP26 efficiently inactivated p38 in vitro.

Furthermore, we tested whether DUSP26 formed a specific physical complex with p38, because docking interactions play important roles in regulating the efficiency and specificity of enzymatic reactions in MAPK cascades (Takekawa et al., 2000; Tanoue et al., 2001). In pull-down assays, when recombinant His-DUSP26 protein, immobilized on Ni–NTA superflow affinity resins, was mixed with lysates from KTA3 cells, endogenous p38 was readily pulled down but His alone was not (Figure 4d). Taken together, p38 is likely to be a direct substrate of DUSP26.

DUSP26 as a critical regulator of p38 MAPK in response to stress in ATC cells

To confirm DUSP26 as an essential negative regulator of the stress-responsive MAPKs, we determined whether this protein would be important for the inactivation of p38 MAPK in distinct stress-responsive paradigms. After KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells were treated with anisomycin, a potent inducer of p38 and JNK (Wu and Bennett, 2005), in different time, activation of p38 MAPK was enhanced in KTA3-DUSP26mt and KTA3-mock cells as compared with KTA3-DUSP26wt cells at the indicated time (Figure 5a). On the other hand, ATC cells transfected with DUSP26-specific siRNA showed increased p38 activation 48 h after anisomycin treatment compared to those transfected with nonspecific control siRNA (Figure 5b). When ATC cell lines with different levels of DUSP26 mRNA expression were treated with different doses of anisomycin, anisomycin-induced early activation of p38 MAPK was significantly attenuated during the first 30 min in ATC cell lines that had shown amplification and overexpression of DUSP26 (8305C and 8505C) compared to cell lines without amplification of DUSP26 (KTA1, KTA3, TCO-1 and TTA2; Figure 5c).

Figure 5
figure5

DUSP26 as a critical regulator of p38 in response to stress in ATC cells. (a) Phospho-specific antibody was used to examine activation of p38 in KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells following treatment with 10 μg/ml of anisomycin for the indicated times. (b) Effect of DUSP26-specific siRNA on activation of p38 in ATC cells. 8305C and 8505C cells were transfected for 48 h with mock, nonspecific control siRNA, or DUSP26-specific siRNA and then stimulated with 10 μg/ml of anisomycin for 30 min. Lysates were collected and submitted to Western blotting with appropriate antibodies. (c) Activation of p38 in ATC cell lines with (8305C and 8505C) or without (KTA1, KTA3, TCO-1 and TTA2) DUSP26 amplification/overexpression after treatment with different doses of anisomycin for 30 min.

DUSP26 as a critical negative regulator of caspase-3-mediated apoptosis

It was conceivable that promotion of cell growth by amplification of DUSP26 in ATC cells could be owing to inhibition of cell death. Indeed, as was shown in Figure 2d, downregulation of DUSP26 by specific siRNA enhanced the degree of cell death as compared with nonspecific control siRNA.

That p38 MAPK is an important regulator of apoptosis has been well documented (Ono and Han, 2000; Chang and Karin, 2001). As DUSP26 inactivates p38, it was reasonable to suggest that the decrease in cell proliferation observed in KTA3-mock and KTA3-DUSP26mt cells could reflect increased sensitivity to apoptosis. To examine this hypothesis, we tested whether DUSP26 is required for regulating apoptosis in response to stress. Anisomycin induces apoptosis via activation of both p38 MAPK and JNK (Wu and Bennett, 2005). As p38 MAPK is hyperactivated in KTA3-mock and KTA3-DUSP26mt cells in response to anisomycin (Figure 5a), we hypothesized that KTA3-mock and KTA3-DUSP26mt cells are likely to be more sensitive to anisomycin-induced apoptosis. Indeed, following anisomycin stimulation, cell death was elevated about twofold in KTA3-DUSP26wt cells, whereas an approximately fourfold increase was observed in both KTA3-DUSP26mt cells and KTA3-mock cells (Figure 6a).

Figure 6
figure6

DUSP26 promotes survival of ATC cells through suppression of caspase-3-mediated apoptosis. (a) KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells were either left unstimulated or stimulated with 2.5 μ M of anisomycin for 12 h to assess anisomycin-induced apoptosis. Dead cells were determined by direct counting using the trypan blue exclusion method. The results are the mean±s.e.m. of three separate experiments. Statistical analysis used the Mann–Whitney U-test: a, KTA3-DUSP26wt cells versus mock-transfected KTA3 cells; b, KTA3-DUSP26wt cells versus KTA3-DUSP26mt cells. All P<0.05. (b) Caspase-3 activity assay. KTA3-DUSP26wt, KTA3-DUSP26mt and KTA3-mock cells were either left unstimulated or stimulated with 2.5 μ M of anisomycin for 12 h. Fold-increases in caspase-3 activity were determined by comparing stimulated cells with unstimulated cells. Statistical analysis used the Mann–Whitney U-test: a, KTA3-DUSP26wt cells versus mock-transfected KTA3 cells; b, KTA3-DUSP26wt cells versus KTA3-DUSP26mt cells. All P<0.05. (c) Caspase-3 activity assay. 8305C cells were transfected with mock, nonspecific control siRNA and DUSP26-specific siRNA for 48 h, and then stimulated with 10 μg/ml anisomycin for 2 h. Fold-increases in caspase-3 activity were determined by comparing stimulated cells with unstimulated cells. The results are presented as the mean±s.e.m. of three separate experiments. Statistical analysis used the Mann–Whitney U-test: a, DUSP26-specific siRNA-transfected cells versus transfection reagent alone-treated (mock) cells; b, DUSP26-specific siRNA-transfected cells versus nonspecific control siRNA-transfected cells. All P<0.05.

The p38 MAPK pathway functions as an upstream mediator of caspase-3 activation, leading to apoptosis via the release of cytochrome c (Wu and Bennett, 2005). To determine whether DUSP26 regulates apoptosis through caspase-3, we tested caspase-3 activity in different cells. KTA3-DUSP26mt and KTA3-mock cells exhibited significantly enhanced levels of caspase-3 activation as compared with anisomycin-treated KTA3-DUSP26wt cells (Figure 6b). On the other hand, 8305C cells treated with DUSP26-specific siRNA exhibited significantly enhanced activation of caspase-3 as compared with nonspecific control siRNA or mock (Figure 6c).

Discussion

The amplification at 8p11–12 has been the subject for many years in various types of cancer, including tumors of breast, urinary bladder, lung and ovary (Theillet et al., 1993; Simon et al., 2001; Ray et al., 2004; Tonon et al., 2005), the actual target oncogene(s) that drive this aberration have never been identified. The sizes and boundaries among the amplicons in these various types of tumor are highly heterogeneous, and different cancers harbor different target genes (Ray et al., 2004; Tonon et al., 2005). Even in the same breast cancer, the target genes may be different among different cells/tissues investigated (Ray et al., 2004; Garcia et al., 2005). Using BAC/PAC-based array-CGH, we defined the amplicon present at 8p12 in some ATC cell lines, and narrowed the gain/amplification to a relatively small region. The SRO in ATC is located within the 8p11–12 amplicon reported in breast-cancer cell lines (Ray et al., 2004), but different from other minimal common amplifications in breast and lung tumors (Garcia et al., 2005; Tonon et al., 2005). Previously proposed candidate genes such as FGFR1, WHSC1L1, TPX2, TACC1, TC-1, FLJ14299, C8orf2, BRF2 and RAB11FIP (Ray et al., 2004; Garcia et al., 2005; Tonon et al., 2005) are present in the region proximal to the ATC amplicon, and the SRO contains five transcripts/genes: FUT10, LOC84549, FLJ23263, DUSP26 and RNF122. Among them, only amplifications of DUSP26 and RNF122 lead to consistent overexpression in ATC cells, strongly suggesting that those two transcripts might be targets for 8p12 gain/amplification in ATC. A recent report showed that DUSP26 was located in the 8p11–12 amplicon of breast-cancer cell lines, and overexpressed in one of three cell lines showing 8p11–12 amplification (Ray et al., 2004).

DUSP26 was overexpressed in ATC cell lines and primary tumor samples. By functional analysis, we showed that exogenously expressed DUSP26 promoted growth of ATC cells, whereas downregulation of endogenous DUSP26 expression by means of siRNA suppressed their growth. Taken together, our results provide strong evidence that DUSP26 shows oncogenic activity in ATC cells and that this gene is the most probable target for amplification in ATC.

DUSP26 is a dual-specificity phosphatase, and its oncogenic action seems to depend on the phosphatase activity. DUSP26 can dephosphorylate both p38 and ERK in ATC cells, but it is more effective at inactivating p38. ERK is thought to be involved in cell proliferation (Widmann et al., 1999), whereas p38 participates in the regulation of apoptosis (Bulavin and Fornace, 2004a), although it has been shown that p38-MAPK cascades enhance survival (Liu et al., 2001; Park et al., 2002), cell growth (Juretic et al., 2001) and differentiation (Yosimichi et al., 2001). Many effectors can alter the balance between ERK and p38, and such changes could have profound consequences for tumor growth and survival.

We investigated whether the promotion of growth of ATC cells caused by amplification of DUSP26 might be owing to inhibition of p38-mediated apoptosis. Indeed, that proved to be so, as downregulation of DUSP26 by transfection with gene-specific siRNA enhanced cell death and p38 activity, in comparison to the effect of nonspecific control siRNA. We also investigated whether growth enhancement in ATC cells was owing to inhibition of caspase-3-mediated apoptosis, because p38 can activate caspase-3. For this experiment, we treated ATC cells with anisomycin that can induce apoptosis via activation of p38 (Wu and Bennett, 2005). Consistent with the enhanced activity of p38 MAPK in response to anisomycin, KTA3-DUSP26mt and KTA3-mock cells exhibited increased levels of apoptosis as compared with KTA3-DUSP26wt cells. By analysing overexpression and gene silencing, we found that DUSP26 could promote growth of ATC cells by inhibiting caspase-3-mediated apoptosis. These data definitively demonstrated that DUSP26 functions as a critical antiapoptotic regulator. Furthermore, downregulation of DUSP26 by siRNA lowered the growth rate compared with nonspecific control siRNA, and this result indicated that downregulation of DUSP26 could also target molecules other than p38 that may negatively contribute to cell-cycle progression, such as cyclin D1 (Casanovas et al., 2000) and Cdc25 phosphatases (Bulavin et al., 2001).

In terms of molecular mechanisms, the Rb and p53 tumor-suppressor proteins are crucial gatekeepers for oncogene-induced transformation in human and rodent cells (Campisi, 1996; Carnero et al., 2002). Oncogene Wip1 phosphatase appears to regulate p16 and p53 activities by regulating p38 (Bulavin et al., 2002, Bulavin et al., 2004b). As p53 is frequently mutated in ATC (Wiseman et al., 2003), DUSP26 might abrogate p16 activity in ATC cells. In our array-CGH analysis, p16 was homozygously deleted in four of the 14 ATC cell lines (TTA1, TTA2, TTA3 and TCO-1; data not shown), indicating that p16 is critical for ATC development. However, in all of the three cell lines with DUSP26 amplification/overexpression (8305C, 8505C and HTC/C3), p16 showed normal DNA copy numbers (data not shown). That observation implied the existence of an additional mechanism for development of ATC, that is, inhibition of p16 activity by DUSP26. Induction of p16 in the presence of oncogenes was reported to be regulated through the mitogen-induced extracellular kinase 1-ERK and p38 pathway (Ohtani et al., 2001; Wang et al., 2002).

Inhibiting positive regulators of cell proliferation, activating tumor-suppressor pathways and inducing apoptosis are primary approaches for intervention in modern cancer therapy. We have shown that overexpression of DUSP26 can promote growth of ATC cells by negatively regulating p38 activity and thereby altering the p38/ERK activity ratio. In addition, suppression of DUSP26 expression significantly inhibited the growth of ATC cells by activating caspase-3. Therefore, DUSP26 could be an ideal therapeutic target for treating primary ATCs.

Materials and methods

Cell lines and primary tumor samples

Fourteen ATC cell lines were employed in this study. Each cell line was maintained in appropriate medium supplemented with 10% FBS and 100 U/ml penicillin/100 μg/ml streptomycin. Seven primary tumor samples were obtained during surgery from patients being treated at the Ito Hospital (Tokyo, Japan). All patients had given informed consent according to guidelines approved by the Institutional Research Board.

Array-CGH analysis

Our in-house MCG Cancer Array-800 (Inazawa et al., 2004) contains 800 BAC/PAC clones carrying genes or sequence-tagged site markers of potential importance in carcinogenesis (http://www.cghtmd.jp/cghdatabase/index.html). Hybridizations were carried out as described elsewhere (Izumi et al., 2005; Takada et al., 2005). Test and reference DNAs were labeled, respectively, with Cy3- or Cy5-deoxycytidine triphosphate. After hybridization, the slides were scanned with a GenePix 4000B (Axon Instruments, Foster City, CA, USA). Acquired images were analysed with GenePix Pro 4.1 imaging software (Axon Instruments).

Fluorescence in situ hybridization

Metaphase chromosomes were prepared from normal male lymphocytes and from ATC cell lines. FISH analyses were performed as described previously (Yu et al., 2003), using BAC clones located in the region of interest as probes.

Reverse transcriptase–polymerase chain reaction

Single-stranded cDNAs were generated from total RNAs, and amplified with primers specific for each gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified to allow estimation of the efficiency of cDNA synthesis. PCR products were electrophoresed in 3% agarose gel. The band quantification was performed using LAS-3000 (Fujifilm, Tokyo, Japan) and Multi Gauge software (Fujifilm). The expression level of DUSP26, normalized with GAPDH, in each sample was divided by that in normal thyroid, and recorded as a ‘fold increase’. All the relevant primer sequences are available on request.

Plasmids

Plasmids expressing Myc-tagged wild-type DUSP26 (pCMV-Tag3-DUSP26wt) and mutant-type (pCMV-Tag3-DUSP26mt) were obtained by cloning the full coding sequences for wild-type DUSP26 and a catalytically inactive mutant form of DUSP26, in which Cys-151 was replaced by Ser (C151S), in-frame into the pCMV-Tag3 vector (Stratagene, La Jolla, CA, USA) along with the Myc-epitope.

For synthesis of recombinant DUSP26 protein, the full coding sequences for wild-type DUSP26 and its catalytically inactive C151S mutant were cloned into pET-23d (+) vector (Novagen, Madison, WI, USA; respectively, pET-23d-DUSP26wt and pET-23d-DUSP26mt).

Colony-formation assays

pCMV-Tag3-DUSP26 or the empty vector (pCMV-Tag3-mock) control were transfected into ATC cells as described previously (Imoto et al., 2003). Expression of DUSP26 protein in transfected cells was confirmed by Western blotting analysis. After 2 weeks of incubation in six-well plates with appropriate concentrations of G418, cells were fixed with 70% ethanol and stained with crystal violet.

Transfection with synthetic siRNA and cell growth assay

An siRNA for DUSP26 was synthesized by Dharmacon Research (Chicago, IL, USA). The target sequence was IndexTermCATCCTTTCCTCAATGTCT (nucleotides 513–531; GenBank accession no. AB158288). A nonspecific control siRNA (D-001206-11-20) was purchased from Dharmacon Research. Each ATC cell line was transfected with 40 nM of synthetic siRNAs using Optifect Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. For measurements of cell growth, the numbers of viable cells were assessed by the microtiter-plate colorimetric WST assay (Cell counting kit-8; Dojindo Laboratories, Kumamoto, Japan; Imoto et al., 2003). For assessing nuclear morphology, cells were fixed with 4% paraformaldehyde, and then stained with 4′,6-diamidino-2-phenylindole.

Purification of 6 × His-tagged protein

BL21-CodonPlus (DE3) cells (Stratagene) were transformed with pET-23d (+)-DUSP26wt and pET-23d (+)-DUSP26mt. Expressed 6xHis-tagged proteins were recovered from cleared E. coli lysates by batch purification using Ni–NTA superflow affinity resin (Qiagen, Hilden, Germany). The recovered proteins were dialysed with 20 mM 2-morpholinoethanesulfonic acid (pH 5.0).

In vitro pNPP phosphatase assay

Activity toward pNPP was measured using pNPP Phosphatase Assay Kits (BioAssay Systems, Hayward, CA, USA) following the manufacturer's protocol. Briefly, pNPP with or without 2–10 μg 6xHis-NATA was incubated in a 96-well microplate at room temperature for 30 min, and the reaction was stopped with a stop solution before its absorbance at 450 nm was measured.

Establishment of clones stably expressing DUSP26 and in vitro proliferation assay

KTA3 cells transfected with pCMV-Tag3B-DUSP26wt, pCMV-Tag3B-DUSP26mt, or empty vector (pCMV-Tag3B-mock) were selected with G418 (0.8 mg/ml) for 3 weeks. For measurements of cell growth, 2 × 103 cells were seeded in 96-well plates. Viable cells were measured by colorimetric WST assay.

Analysis of MAP kinase activities

KTA3-DUSP26wt, KTA3-DUSP26mt or KTA3-mock cells were deprived of serum for 24 h and stimulated with 10% FBS for 30 min. The cells were either left untreated or stimulated with 10 μg/ml anisomycin for 0.5, 1, 2 or 4 h. Each culture was transfected with mock, DUSP26-specific siRNA or nonspecific control siRNA for 48 or 72 h as described above, and then either left untreated or stimulated with 10 μg/ml anisomycin for 30 min. To check the activity of p38, the cells were treated with various doses of anisomycin for 30 min, and lysed in buffer (50 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraaceticacid (EDTA), 0.5% Nonidet P-40, 2 mM Na3VO4, 100 mM NaF, 10 mM sodium diphosphate decahydrate) supplemented with 5 μg/ml leupeptin, 5 μg/ml aprotinin and 1 μg/ml pepstatin. Equivalent amount of cell lysates were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and immunoblotted with anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-SAPK/JNK (Cell Signaling Technologies, Danvers, MA, USA), anti-phospho-JNK or anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The band quantification was performed using LAS-3000 (Fujifilm) and Multi-Gauge software (Fujifilm).

In vitro dephosphorylation reactions

KTA3-DUSP26wt and KTA3-DUSP26mt cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.4. 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholic acid) supplemented with 5 μg/ml leupeptin, 5 μg/ml aprotinin and 1 μg/ml pepstatin, then treated at 30°C for 2 h in phosphatase buffer (50 mM Tris-HCl, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 0.01% Brij 35, 2 mM MnCl2, pH 7.5) with 1000 U of λ-protein phosphatase (New England Biolabs, Beverly, MA, USA).

To prepare phosphorylated p38, KTA3 cells were stimulated with 10 μg/ml anisomycin for 30 min and lysed in RIPA buffer. Activated p38 proteins were immunoprecipitated and incubated at 30°C for 2 h with phosphatase buffer and purified 6xHis-DUSP26wt or 6xHis-DUSP26mt. The phosphorylation status of p38 was monitored by immunoblotting with anti-phospho-p38 antibody.

In vitro binding assays

BL21 cells carrying His or His-DUSP26 were grown in Luria–Bertani medium and lysed. The lysates were incubated for 1 h at 4°C with Ni–NTA superflow affinity resins. KTA3 cells were lysed with RIPA buffer after treatment with anisomycin 10 μg/ml for 30 min because phosphatases preferentially bind phosphorylated and activated substrates (Takekawa et al., 2000). The lysates of KTA3 cells were mixed with Ni–NTA superflow affinity resins immobilized with His fusion proteins at 4°C for 2 h. Proteins were eluted with 1 × sample buffer, subjected to SDS–PAGE, and immunoblotted with anti-p38 antibody.

Assays for apoptosis and caspase-3 activity

Cells were either left untreated or treated with 2.5 μ M anisomycin for 12 h. Adherent and non-adherent cells were collected, and cell death was determined by counting the number of trypan blue-positive and -excluded cells. The percentage of dead cells was quantified by normalizing the number of trypan blue-positive cells to the total number of cells present. Caspase-3 activity was measured by Caspase-3/CPP32 Colorimetric Assay Kit (Medical & Biological Laboratories, Nagoya, Japan) according to the manufacturer's instructions.

Accession codes

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Acknowledgements

We are grateful to Professor Y Nakamura (Human Genome Center, Institute of Medical Science, The University of Tokyo) for continual encouragement throughout this work. This work was supported by grants-in-aid for Scientific Research (to J Inazawa, I Imoto) and Scientific Research on Priority Areas (C) (to J Inazawa, I Imoto), and a 21st Century Center of Excellence (COE) Program for Molecular Destruction and Reconstruction of Tooth and Bone (to J Inazawa, W Yu) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and a grant from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST) (to J Inazawa). W Yu is a Heiwa Nakajima Foundation Scholar.

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Yu, W., Imoto, I., Inoue, J. et al. A novel amplification target, DUSP26, promotes anaplastic thyroid cancer cell growth by inhibiting p38 MAPK activity. Oncogene 26, 1178–1187 (2007) doi:10.1038/sj.onc.1209899

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Keywords

  • DUSP26
  • ATC
  • amplification
  • p38
  • array-CGH

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