Oncogene (2008) 27, 5684–5695 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Roles of induced expression of MAPK -2 in tumor development in RET-MEN2A transgenic mice

T Hasegawa1,2, A Enomoto1,3, T Kato1, K Kawai1, R Miyamoto1, M Jijiwa1, M Ichihara4, M Ishida1, N Asai1, YMurakumo 1, K Ohara1,2, YNiwa 2, H Goto2 and M Takahashi1,5

1Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Gastroenterology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3Institute for Advanced Research, Nagoya University, Nagoya, Japan; 4Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Japan and 5Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan

Germline mutations in the RET tyrosine kinase gene are line-derived neurotrophic factor (GDNF) family responsible for the development of multiple endocrine of ligands (GFLs). It has a crucial role in transducing neoplasia 2A and 2B (MEN2A and MEN2B). However, growth and differentiation signals in tissues knowledge of the fundamental principles that determine derived from the neural crest and the developing kidney the mutant RET-mediated signaling remains elusive. (Schuchardt et al., 1994; Moore et al., 1996; Pichel Here, we report increased expression of -activated et al., 1996; Sa´ nchez et al., 1996; Treanor et al., 1996; phosphatase-2 (MKP-2) in carcinomas Rosenthal, 1999; Airaksinen and Saarma, 2002). developed in transgenic mice carrying RET with the It has been firmly established that germline mutations MEN2A mutation (RET-MEN2A). The expression of in the RET gene are responsible for several forms of MKP-2 was not only induced by RET-MEN2A or RET- human diseases. RET loss-of-function mutations MEN2B mutant proteins but also by the activation of predispose for enteric neural crest cell dysfunction, endogenous RET by its ligand, glial cell line-derived which leads to Hirschsprung’s disease, whereas its gain- neurotrophic factor (GDNF). MKP-2 expression was also of-function mutations are found in several human evident in the MKK-f cell line, which was established from cancers, including multiple endocrine neoplasia (MEN) a mammary tumor developed in a RET-MEN2A trans- type 2A (MEN2A) and 2B (MEN2B), familial medul- genic mouse. Inhibition of MKP-2 attenuated the in vitro lary thyroid carcinoma (FMTC) and papillary thyroid and in vivo proliferation of MKK-f cells, which was carcinoma (Donis-Keller et al., 1993; Mulligan et al., mediated by the suppression of B1 expression. 1993; Carlson et al., 1994; Edery et al., 1994; Hofstra Furthermore, we found that MKP-2 is highly expressed in et al., 1994; Romeo et al., 1994). The MEN2A medullary thyroid carcinomas derived from MEN2A mutations were identified primarily in cysteine residues patients. These findings suggest that the increased of the RET extracellular domain that lead to ligand- expression of MKP-2 may play a crucial role in oncogenic independent RET dimerization via the formation signaling downstreamof mutant RET, leading to dereg- of an intermolecular disulfide bond (Asai et al., 1995; ulation of . Santoro et al., 1995; Eng, 1999). The MEN2B muta- Oncogene (2008) 27, 5684–5695; doi:10.1038/onc.2008.182; tions, characterized by a methionine-to- published online 9 June 2008 change at codon 918 or an alanine-to-phenylalanine change at codon 883 in the tyrosine kinase domain, Keywords: RET; MEN2A; MKP-2; cell proliferation; result in an increase of intrinsic RET catalytic activity cell cycle (Carlson et al., 1994; Gimm et al., 1997; Smith et al., 1997). Of particular interest are marked differences in affected organs and disease phenotype among indivi- duals and kindreds with MEN2A, MEN2B or FMTC Introduction mutations (Kodama et al., 2005; Asai et al., 2006). MEN2A is characterized by MTC, pheochromocytoma The RET proto-oncogene encodes a receptor tyrosine and parathyroid adenoma, whereas MEN2B shows a kinase that acts as a functional receptor for the glial cell more complex phenotype, with association of MTC, pheochromocytoma, mucosal neuroma, intestinal gang- lioneuromatosis and skeletal deformity. FMTC patients Correspondence: Professor M Takahashi, Department of Pathology, develop MTC only. The diversity and complexity of the Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, phenotypes observed with MEN2A, MEN2B and Showa-ku, Nagoya, Aichi 466-8550, Japan. E-mail: [email protected] FMTC patients suggest that the mutant RET protein Received 8 January 2008; revised 14 April 2008; accepted 7 May 2008; is responsible for distinct patterns of intracellular published online 9 June 2008 signaling cascades. Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5685 Previous studies have revealed that constitutive To define the genes that are potentially involved in phosphorylation of tyrosine residues in the intracellular tumorigenesis in RET-MEN2A transgenic mice, we domains of RET-MEN2A and RET-MEN2B mutant compared gene expression between pooled carcinomas proteins results in activation of the Ras/Raf/mitogen- and normal tissues from six independent mice using activated protein kinase (MAPK), phosphatidylinositol- cDNA microarrays (Table 1). The expression levels of 3 kinase/Akt, p38 MAPK and c-Jun amino-terminal 11 genes were upregulated more than twofold (log ratio) kinase (JNK) pathways (Takahashi, 2001; Airaksinen in all of the carcinoma tissues (thyroid, salivary and and Saarma, 2002; Kodama et al., 2005; Asai et al., mammary carcinomas). Validation of the microarray 2006). Because all of these pathways are also activated results with quantitative reverse transcription (RT)-PCR by the GFLs via endogenous RET, the question arises as showed that 7 of the 11 genes showed similar trends or to how the signals downstream of the RET mutants are much higher expression levels when compared with the distinct from those promoted by normal RET, in which microarray data. These seven genes were mouse Ret, the duration and strength of signals are tightly Col11a1, Cxcl1, Usp18, Slc35d3, MKP-2 and Tde2l regulated. (Table 1). To elucidate the developmental mechanisms of the MEN2A phenotype, we established transgenic mice expressing RET-MEN2A that developed salivary, thyr- Induction of MKP-2 expression by RET-MEN2A, oid and mammary gland carcinomas (Kawai et al., RET-MEN2B or normal RET activation 2000). In this study, cDNA microarrays were used to MKP-2 (also termed DUSP4), a dual-specificity phos- analyse global gene expression patterns in salivary, phatase that possesses phosphatase activity toward thyroid and mammary carcinomas to identify potential MAPKs (p42/44 MAPK, p38 MAPK and JNK) molecular determinants of RET-MEN2A-dependent (Farooq and Zhou, 2004; Dickinson and Keyse, 2006; tumorigenesis. MAPK phosphatase-2 (MKP-2; Owens and Keyse, 2007) was chosen for further study. DUSP4), one of the genes that was upregulated in all The expression levels of the MKP-2 protein were the carcinomas was chosen for further study. MKP-2 examined by immunostaining sections from the mam- was first identified as a dual-specificity phosphatase that mary and salivary carcinomas and normal tissues from selectively dephosphorylates the p42/44 MAPK, p38 RET-MEN2A transgenic mice (Figure 1a). Consistent MAPK and JNK (Guan and Butch, 1995; Misra-Press with the microarray analysis, MKP-2 expression was not et al., 1995; Farooq and Zhou, 2004; Cadalbert et al., detected in the ductal epithelial cells of the normal 2005; Dickinson and Keyse, 2006; Owens and Keyse, mammary and salivary glands but was highly expressed 2007). Interestingly, despite the activity of MKP-2 in in the mammary and salivary carcinomas. MKP-2 inhibiting the activity of the MAPKs, depletion of immunoreactivity was detected predominantly in the MKP-2 by RNA interference resulted in deregulation of cytoplasm and weakly in the nucleus. the cell cycle and proliferative inhibition of MKK-f, a We next examined whether the expression of MKP-2 cell line that was established from a mammary is induced by the overexpression of mutant RET carcinoma developed in a RET-MEN2A transgenic protein. MKP-2 transcript and protein expression were mouse (Kawai et al., 2003). We also found that the significantly upregulated in NIH3T3 cells that stably expression of MKP-2 was induced downstream of expressed RET-MEN2A or RET-MEN2B (Figures 1b GDNF activation of normal RET in a temporally and c). MEF3T3 lines that express RET-MEN2A and regulated manner that may orchestrate the tempo and RET-MEN2B under the control of a tetracycline- duration of MAPK activation in a physiological suppressible (Tet-off) promoter were also used to context. These data suggest that constitutively induced examine the effect of mutant RET on MKP-2 expres- expression and activity of MKP-2 may be necessary to sion. At 2 days after the induction of RET, the levels of maintain continuous proliferation of RET-MEN2A- MKP-2 mRNA and protein were analysed by semi- driven carcinomas and present a previously unrecog- quantitative RT–PCR and western blot analysis, respec- nized role for MKP-2. tively. As shown in Figures 1d and e, MKP-2 mRNA and protein were induced over a time course similar to that of RET induction. The expression of MKP-2 was also apparent in the MKK-f cell line, which was Results established from mammary carcinoma from a RET- MEN2A transgenic mouse (Kawai et al., 2003) (Figures Analysis of gene expression in carcinomas from 1b and c). These results indicate that mutant RET is RET-MEN2A transgenic mice capable of inducing MKP-2 expression. We previously reported the establishment of RET- To investigate whether MKP-2 expression is induced MEN2A transgenic mice, which express a recombinant by the activation of endogenous RET by GDNF, TGW human RET-MEN2A gene (Cys634-Arg) driven by neuroblastoma cells, which have been shown to express Moloney murine leukemia virus long terminal repeat both endogenous RET and GFRa1 (Watanabe et al., (MoMuLV LTR) (Kawai et al., 2000). At 6 months of 2002; Uchida et al., 2006), were treated with GDNF. age, all transgenic mice developed MTC and approxi- MKP-2 was weakly expressed in TGW cells in their mately one-half of the mice developed salivary gland or basal state, and biphasic induction of MKP-2 was mammary gland adenocarcinomas (Kawai et al., 2000). observed 15 min and 6 h after GDNF stimulation

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5686 (Supplementary Figure S1). The biphasic induction of MKP-2 was transient, thus it is conceivable, in physiological contexts, that the temporally regulated Q-PCR expression of MKP-2 may orchestrate the duration and (fold change) strength of the MAPK activation in endogenous RET activation. Though the first phase of MKP-2 upregula- tion was correlated with consequent of p42/44 MAPK, its second phase, which reached a maximum at 6 h after GDNF stimulation, did not TG mammary carcinoma WT mammalian glands vs apparently correlated with the re-activation and depho- cDNA array sphorylation of p42/44 MAPK. The data may hint at (signal log ratio) more complicated function of MKP-2 that is not limited

ammary carcinomas from RET-MEN2A to the inhibition of MAPKs activation. imilar results were obtained. The signal log Gene symbol and name are listed. Data of

MKP-2 exhibits phosphatase activity toward p42/44 Q-PCR and p38 MAPKs,but not JNK,in MKK-f cells (fold change) Although previous studies have shown that MKP-2, when activated by p42/44 MAPK, possesses the activity to dephosphorylate p42/44 and p38 MAPKs, and JNK, its substrate specificity remains controversial (Farooq WT thyroid glands vs TG thyroid carcinoma and Zhou, 2004; Cadalbert et al., 2005; Dickinson and Keyse, 2006; Owens and Keyse, 2007). To confirm the cDNA array

(signal log ratio) phosphatase activity of MKP-2, HEK293 cells were transfected with mouse wild-type MKP-2 and its catalytically inactive mutant (MKP-2 C280S), in which the cysteine at residue 280 in the catalytic domain was mutated to serine (Cadalbert et al., 2005). Epidermal

Q-PCR growth factor (EGF)-mediated phosphorylation of p42/

(fold change) 44 MAPK was attenuated by exogenous expression of wild-type MKP-2 and enhanced by the C280S mutant (Figure 2a). No difference was observed in the activa- tion of p38 MAPK and JNK between wild-type MKP-2 and CS mutant-transfected HEK293 cells (data not TG salivary carcinoma shown). cDNA array The effect of RET-MEN2A-induced MKP-2 expres- (signal log ratio) sion on the activity of MAPKs was further examined by the small-interfering RNA (siRNA)-mediated transient depletion (knock down) of MKP-2 in MKK-f cells. The activation of p42/44 and p38 MAPKs was modestly augmented by the depletion of MKP-2 in MKK-f cells (Figure 2b). In contrast, there was little effect on the activation of JNK, suggesting that MKP-2 possesses phosphatase activity toward p42/44 and p38 MAPKs in MKK-f cells. These findings, in conjunction with those from the HEK293 cells, suggest that the effects of phosphatase activity of MKP-2 on MAPKs differ depending on the cell line used. On the whole, the effect of MKP-2 depletion on the dephosphorylation of the A list of genes upregulated in salivary, thyroid and mammary gland carcinomas developed in RET-MEN2A transgenic mice MAPKs was not remarkable, which may be accounted for by the fact that other members of MKPs, especially MKP-3 (DUSP6) and MKP-4 (DUSP9), were upregu- Table 1 lated in MKP-2-depleted MKK-f cells (Supplementary Figure S2).

Depletion of MKP-2 inhibits the proliferation of MKK-f cells in vitro and in vivo We next investigated the effect of MKP-2 on the proliferation of RET-MEN2A-driven cancer cells. Two lines of MKK-f cells were established in which MKP-2 Gene symbol Gene assignment WT salivary gland vs Ret (mouse)Col11a1D11Lgp2e Ret proto-oncogeneCxcl1Ifitl Collagen, DEXH type (Asp-Glu-X-His) XI,Usp18 box alpha polypeptide 1 58D930024E11Irf7 Chemokine DNA (C-X-C segment, motif)Slc35d3 Chr ligand 9, 1 ERATOMKP-2 Ubiquitin-specific Doi Interferon-induced peptidase 280, protein 18 expressed withTde2l tetratricopeptide repeats 1 Solute carrier InterferonAbbreviations: family regulatory RT, 35, Dual-specificity factor reverse member phosphatase 7 transcription; D3transgenic 4 TG, mice RET-MEN2A and normal transgenic tissuesratio mice; from Tumor WT, indicates wild-type differentially wild-type fold mice. expressed 3.0 mice. change Thequantitative 2-like data Note: presented 3.5 RT–PCR of microarray in (Q-PCR) quantitative analysis logarithmic RT–PCR are of form. are shown salivary, representative Genes with of thyroid upregulated comparison 2.8 two and more to independent m than experiments wild-type twofold in tissues. which (log s ratio) in all of the carcinomawas tissues are shown. 1.69 2.44 1.2 3.3 depleted 7.0 34 2.8 3.7 by 2.0 10 3.6 39.2stable 000 2.2 2.8 2.6 231 2.6 1132expression 3.47 2.59 4.6 3.46 2.0 917 842 2.6 1.1of 1.61 2.7 short-hairpin 5.0 2.4 12.1 2.2 2.4 2.3 3.87 2.3 2.0 2.7 2.07 RNA 9.7 5.19 3.3 1.92 2.0 3.12 397.4 3.3 3.3 22.5 2.8 6.0 448 11.85 3.0 2.0 69 2.4 18.47 40.72 84.6 9.9 126

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5687 IS: anti-MKP-2

Mammary gland Mammary carcinoma

NIH3T3 NIH3T3-2ANIH3T3-2BMKK-f Case 1 Case 2 Case 3 MKP-2

β-actin

Salivary gland Salivary carcinoma NIH3T3 NIH3T3-2ANIH3T3-2BMKK-fMr (kDa) Case 1 Case 2 IB: RET51 -175

IB: MKP-2 -47.5

IB: β-actin -47.5

RET-MEN2A RET-MEN2B Mr (kDa) IB: RET51 -175 Tet (+)Tet (-) Tet (+) Tet (-)

IB: MKP-2 -47.5 Day 0 Day 1 Day 2 Day 0 Day 1 Day 2 β MKP-2 IB: -actin -47.5

5 10 (AU) (AU) 1 1 Fold change Fold change Figure 1 Induced expression of mitogen-activated protein kinase phosphatase (MKP)-2 by multiple endocrine neoplasia 2A and 2B mutant RET (RET-MEN2A and RET-MEN2B). (a) The expression of MKP-2 in mammary (upper panels) and salivary (lower panels) carcinomas derived from RET-MEN2A transgenic mice. Sections from mammary and salivary normal gland and carcinomas derived from RET-MEN2A transgenic mice were stained with anti-MKP-2 antibody. Representative data of immunohistochemistry using four normal and three carcinoma tissues from independent transgenic mice were shown. Arrows denote normal epithelial cells. The region within the black box is shown at a higher magnification in the inset. (b, c) Induction of MKP-2 expression by RET- MEN2A and RET-MEN2B. Total RNA and cell lysates from control NIH3T3 cells, NIH3T3 cells stably expressing RET–MEN2A (Cys634-Arg) or RET-MEN2B (Met918-Thr), and MKK-f cells were extracted and analysed by reverse transcription (RT)–PCR (b) and western blotting (c). IB, immunoblotting. (d, e) MEF3T3 Tet-off cells were stably transfected with pTRE expression vectors carrying RET-MEN2A or RET-MEN2B cDNA. Total RNA and cell lysates from cells 24 and 48 h after tetracycline withdrawal were analysed by RT–PCR (d) and western blotting (e). Graphs show the quantification of intensity of MKP-2 bands in the RT–PCR and western blots. Tet, tetracycline (doxycycline); AU, arbitrary units.

(shRNA) targeting MKP-2 (Figures 3a and b). The inoculated subcutaneously with MKP-2-depleted or morphology of MKP-2-depleted MKK-f cells took the control MKK-f cells (3 Â 106), and the size and weight form of a sarcomatoid pattern, in contrast to control of tumors were measured for 4 weeks (Figure 4). Tumor cells that exhibited a polygonal epithelioid appearance growth of MKP-2-depleted cells was significantly (Figure 3b). As shown in Figure 3c, the proliferation attenuated compared with control cells (Figures 4a rate of MKP-2-depleted cells was significantly lower and b), indicating that MKP-2 expression promotes than that of control cells. However, despite the changes proliferation of RET-MEN2A-driven cancer cells of morphological and biological phenotypes, no obvious in vivo. difference of the MAPK phosphorylation was observed between control and MKP-2 knockdown cells (Figure 3d), which further suggests that other members Depletion of MKP-2 induces cell cycle delay in the G2/M of MKPs may compensate for the loss of MKP-2. phase through downregulation of cyclin B1 The subcutaneous injection of MKK-f cells into nude To observe closely the effects of MKP-2 depletion on mice was performed to ascertain the in vivo role of the proliferation of MKK-f cells, the distribution of cells MKP-2 in tumor growth. Each group of six mice was in different phases of the cell cycle was analysed by flow

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5688

siRNA: Control MKP-2 Mr (kDa)

MKP-2-myc IB: MKP-2 -47.5 Vector WT CS IB: pp42/44 MAPK -47.5 EGF (100 ng/ml) -+-- + +Mr (kDa) -47.5 -47.5 IB: pp42/44 MAPK IB: p42/44 MAPK

IB:pp38 MAPK -47.5 IB: p42/44 MAPK -47.5

IB: p38 MAPK -47.5 IB: MKP-2 -47.5 IB: pJNK -62 IB: β-actin -62 IB: JNK -62 HEK293 cells IB: β-actin -47.5 MKK-f cells Figure 2 Mitogen-activated protein kinase phosphatase (MKP-2) possesses phosphatase activity toward MAPKs in HEK293 and MKK-f cells. (a) HEK293 cells transfected with control vector, MKP-2-myc (WT) or the inactive MKP-2 CS mutant (C280S) were serum-starved and incubated with epidermal growth factor (EGF) (100 ng/ml) for 15 min. The cell lysates were analysed by western blotting using the indicated antibodies. The activation of p42/44 MAPK was significantly suppressed in cells expressing MKP-2 (WT) and enhanced by the CS mutant. (b) The effects of small-interfering RNA (siRNA)-mediated depletion of MKP-2 on the activation of MAPKs in MKK-f cells. Total cell lysates from control or MKP-2 siRNA-transfected MKK-f cells were subjected to western blot analysis using the indicated antibodies. Phosphorylation of p42/44 MAPK and p38 MAPK was increased by the transfection of MKP-2 siRNA.

cytometry (Figure 5a). MKP-2-depleted and control tional inhibition rather than protein degradation MKK-f cells were synchronized at the G1/S transition (Figure 5d). by thymidine treatment for 24 h, harvested at the Cyclin B1 is the cyclin that binds to and activates cdc2 indicated time points, and analysed by flow cytometry. kinase (also termed CDK1) (Takizawa and Morgan, Cells depleted in MKP-2 suffered a significant delay at 2000; Porter and Donoghue, 2003). As predicted, in the the G2/M transition (Figure 5a, lower panel), implying MKP-2-depleted MKK-f cells, downregulation of cyclin that MKP-2 depletion prevents proliferation by B1 resulted in a decrease in cdc2 kinase activity in both either blocking the cell cycle or delaying mitosis. asynchronized and synchronized MKK-f cells (Figures Immunostaining analyses also revealed that MKP-2 5b and e). Thus, the inhibition of cell proliferation by knockdown causes an accumulation of mitotic MKP-2 depletion appears to be associated with down- cells, especially at prophase and prometaphase, in regulation of cyclin B1, which results in the inhibition of which the chromosomes start to condense and separa- cdc2 kinase activity and cell cycle delay in the G2/M tion of the centriole pairs was completed, respectively transition. The downregulation of cyclin B1 was also (Supplementary Figure S3). confirmed by immunocytochemistry, in which the The molecular mechanism of the G2/M blockade appearance of cyclin B1 on the spindle poles at mediated by the depletion of MKP-2 was examined via prometaphase was decreased in MKP-2-depleted expression of cell cycle regulators, including those MKK-f cells compared with control cells (Figure 5f). implicated in the G2/M transition (Figure 5b). The It seems that MKP-2 exerts its regulatory effects during depletion of MKP-2 in MKK-f cells resulted in up to the cell cycle independent of the de-phosphorylating eightfold suppression of cyclin B1 expression, a regula- activity of MAPKs, because forced activation of tory subunit of M-phase-promoting factor that has a p42/44 MAPK or p38 MAPK did not lead to the crucial role in the G2/M checkpoint (Norbury and downregulation of cyclin B1 (Supplementary Figures Nurse, 1992; Pines, 1999; Takizawa and Morgan, 2000; S4a and b). Porter and Donoghue, 2003). In contrast, we did not observe any effects on the expression of other and cyclin-dependent kinases (CDKs) that were exam- Expression of MKP-2 and cyclin B1 in ined. The downregulation of cyclin B1 expression was MEN2A-associated MTCs mediated by both shRNA and siRNA-mediated deple- Finally, to test whether the expression of MKP-2 is tion of MKP-2 (Figures 5b and c), demonstrating that induced in human hereditary cancers driven by RET the downregulation of cyclin B1 is not attributed to ‘off- mutations, the expression of MKP-2 was examined in target’ effects of RNAi. The downregulation of cyclin human MTC specimens. MKP-2 was highly expressed in B1 in MKP-2-depleted cells was mediated by transcrip- MTC tumor cells, from five MEN2A patients, in which

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5689 Control shRNA

∗ Clone 3 5 ∗ Clone 4

4

shRNA: Control MKP-2 3

MKP-2 MKP-2 shRNA

[Absorbance] 2 Clone 20

1 Clone 24 β-actin

MKK-f cells 0 0 123 [Day]

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Control shRNA

Clone 3 Clone 4 Clone 3 Clone 4 Clone 20 Clone 24 IB: MKP-2

IB: pp42/44 MAPK

MKP-2 shRNA IB: p42/44 MAPK

Clone 20 Clone 24 IB: pp38 MAPK

IB: p38 MAPK

IB: β-actin

MKK-f cells Figure 3 The effects of mitogen-activated protein kinase phosphatase (MKP)-2 depletion on morphology and proliferation of MKK-f cells. (a) Short-hairpin RNA (shRNA)-mediated depletion of MKP-2 in MKK-f cells. MKK-f cells were transduced with empty (control) retroviral vectors or with vectors harboring MKP-2 shRNA. Total RNA was analysed by RT–PCR. (b) The effect of MKP-2 depletion on cell morphology. After transduction with retroviral vectors, MKK-f clones were selected in which the expression of MKP- 2 was effectively suppressed. Phase contrast images of control (clones 3 and 4) or MKP-2 shRNA-transduced MKK-f cells (clones 20 and 24) are shown. (c) The effect of MKP-2 depletion on the proliferation of MKK-f cells. The proliferation rates of MKK-f cells transduced with control (clones 3 and 4) or MKP-2 shRNA (clones 20 and 24) were examined by WST-1 assays as described in the Materials and methods. Data are presented as means±s.d. The proliferation of MKP-2-depleted cells was significantly impaired compared with control cells (*Po0.05). (d) The effect of stable depletion of MKP-2 on the activity of p42/44 and p38 MAPKs. Total cell lysates from MKK-f cells transduced with control or MKP-2 shRNA were analysed by western blotting using anti-pp42/44 and pp38 MAPK antibodies. the overexpression of cyclin B1 was also demonstrated Discussion (Figure 6a). Immunofluorescent studies showed that MKP-2 is apparently expressed in the cytoplasm, with Germline or somatic RET mutations lead to various weak expression in the nucleus, of tumor cells that were forms of human diseases. Its gain-of-function mutations positive for calcitonin, an MTC marker (Figure 6b). are responsible for MEN2A, MEN2B and FMTC, as Weak MKP-2 expression was also detected in parafolli- well as sporadic MTC and papillary thyroid carcinoma. cular C cells in MEN2A patients (Figure 6c), whereas it Despite much investigation, it is still unknown was very low or undetectable in normal thyroid how signaling downstream of the oncogenic RET is follicular and parafollicular cells (Figures 6b–e). distinct from that mediated by normal RET activation

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5690

p < 0.05 Control shRNA ∗ ) MKP-2 shRNA

3 1200 1.2 ∗ 1 ∗ 800 0.8

0.6

400 0.4 Volume of tumors (mm Volume Weight of tumors (g) Weight 0.2

0 0 1 234 Control MKP-2 Week shRNA Figure 4 Inhibition of mitogen-activated protein kinase phosphatase (MKP)-2 attenuates the growth of MKK-f cells in vivo. (a) Control or MKP-2-depleted MKK-f cells (3 Â 106) were subcutaneously injected into two groups of nude mice. Each group, one control and one experimental, was comprised of six mice. The volumes of primary tumors were measured at the indicated time points after injection. The mean tumor size±s.d. was determined for both groups of mice. The tumor growth of MKP-2-depleted cells was significantly impaired compared with control cells (*Po0.05). (b) At 4 weeks after the injection, the weights of the primary tumors were measured. The weight of MKP-2-depleted tumors was significantly lower than that of the control tumors (Po0.05).

(Airaksinen and Saarma, 2002; Kodama et al., 2005; expression of MKP-2 suppresses the phosphorylation of Asai et al., 2006). MAPKs and elevates cellular apoptotic response. We The present study was designed to gain insights into also examined whether the depletion of MKP-2 influ- the mechanisms underlying the action of mutant RET ences of MKK-f cells by oxidative stress or protein. DNA microarray analysis was used to identify cisplatin, an anticancer drug. However, no significant differentially expressed genes relevant for tumor devel- difference in apoptosis was observed between MKP-2- opment in RET-MEN2A transgenic mice. We found depleted and control cells (data not shown). The that the expression of MKP-2 is significantly induced discrepancies between previous reports and our present in RET-MEN2A-driven carcinoma cells and plays an data may be due to differences in cellular sensitivity important role in their proliferation both in vitro against oxidative stress and anticancer reagents. and in vivo. MKP-2 is a dual-specificity phosphatase Elucidation of the fundamental principles that deter- known to inactivate MAPKs (Farooq and Zhou, 2004; mine how the induction and/or activation of MKP-2 Cadalbert et al., 2005; Dickinson and Keyse, 2006; Owens results in distinct biological outcomes requires further and Keyse, 2007), as was confirmed by the present study investigation. (Figure 2). However, despite an important function of In spite of the remarkable success of MKP-2 knock- MKP-2 in regulating the activity of MAPKs, evidence is down, there is little, if any, effect on the depho- limited for its role in tumor progression. In contrast to sphorylation of the MAPKs in MKK-f cells, which what was speculated, RNAi-mediated stable depletion may be accounted for by compensation mechanisms or of MKP-2 resulted in a decrease in the proliferation of suggests that the function and substrate specificity of MKK-f cells, suggesting a possible role of MKP-2 in MKP-2 may be much more complicated than previously potentiating tumorigenesis. The underlying mechanisms described. An insight gained from this study is the for this finding are currently unknown. Because involvement of MKP-2 in the regulation of cyclin B1 continuous overactivation of Ras/Raf/MAPK signaling expression and cdc2 kinase activity, both of which in some contexts can result in senescence, cell cycle are required for G2/M progression of the cell cycle arrest and/or apoptosis (Sewing et al., 1997; Woods (Norbury and Nurse, 1992; Pines, 1999; Porter and et al., 1997; Lundberg et al., 2000; Murphy and Blenis, Donoghue, 2003). Increasing evidence indicates that 2006), MKP-2 might be required for orchestrating the deregulation and overexpression of cyclin B1 is involved strength and duration of MAPK signaling to potentiate in neoplastic transformation in many tumor types (Soria tumor growth and survival. As observed for endogenous et al., 2000; Hassan et al., 2002; Yuan et al., 2006). RET activation in the presence of its ligand GDNF and Constitutive activation of cyclin B1 and associated cdc2 its coreceptor GFRa1 (Supplementary Figure S1), kinase can override the G2 DNA damage checkpoint, transient, but not constitutive, expression of MKP-2 which leads to an accumulation of genomic defect found could be essential for regulated proliferation in physio- in malignant tumors (Kao et al., 1997; Taylor et al., logical conditions. 1999; Park et al., 2000). Recent reports that forced Previous studies have reported that the expression of RNAi-mediated reduction of cyclin B1-induced contin- MKP-2 is induced through p53 or E2F-1 transcription uous apoptosis in tumor cells (Yuan et al., 2004, 2006) factors following oxidative stress (Shen et al., 2006; are in accordance with this observation. Thus, MKP-2- Wang et al., 2007). In this context, exogenous over- mediated expression of cyclin B1 may be important for

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5691

0 h 3 h 6 h 9 h 12 h -2 P shRNA: ontrol K Control shRNA C M Mr (kDa) -2 (clone 4) IB: cyclin B1 -62 ontrol KP

Cell count siRNA: C M DNA content IB: cdc2 -32.5 G1 23% G1 22% G1 41% G1 29% IB: p-cdc2 -32.5 S 54% S 14% S 27% S 33% IB: cyclin B1 G2/M 22% G2/M 64% G2/M 31% G2/M 34% IB: β-actin -47.5

0 h 3 h 6 h 9 h 12 h IB: cyclin A -47.5

IB: cyclin D1 -32.5 ontrol MKP-2 shRNA shRNA: C MKP-2 (clone 20) IB: cyclin E -47.5 cyclin B1 Cell count IB: CDK2 -32.5 DNA content β-actin G1 25% G1 25% G1 23% G1 38% IB: CDK4 -32.5 S 58% S 29% S 26% S 28% G2/M 16% G2/M 45% G2/M 49% G2/M 29% IB: CDK6 -47.5

Control shRNA (clone 4) Control shRNA (clone 4) MKP-2 shRNA (clone 20) IB: cyclin B1

IB: cdc2

IB: p-cdc2 03 6912(h) DAPILamin A/C DAPI Lamin A/C MKP-2 shRNA (clone 20) IB: cyclin B1

IB: cdc2

IB: p-cdc2 cyclin B1Merged cyclin B1 Merged 036912(h) (Prometaphase) Figure 5 Depletion of mitogen-activated protein kinase phosphatase (MKP)-2 induces G2/M delay through the inhibition of cyclin B1 expression in MKK-f cells. (a) Control (upper panel) or MKP-2-depleted (lower panel) MKK-f cells were synchronized at the G1/S transition with a double-thymidine block and released to fresh medium. Cells were harvested at the indicated time points and cell cycle analyses were performed using a flow cytometer. The experiments were repeated three times and a representative histogram is shown. The cell population in each cell cycle stage is indicated under the histogram. (b) Total cell lysates of control or MKP-2-depleted MKK-f cells were analysed by western blotting using the indicated antibodies. CDK, cyclin-dependent kinase. (c) Total cell lysates of control or MKP-2 small-interfering RNA (siRNA)-transfected MKK-f cells were analysed by western blotting using anti-cyclin B1 antibody. (d) Total RNA of control or MKP-2-depleted MKK-f cells was analysed by RT–PCR using cyclin B1-specific primers. (e) Control (upper panel) or MKP-2-depleted (lower panel) MKK-f cells were synchronized at the G1/S transition and released to fresh medium. Total cell lysates harvested at the indicated time points were subjected to western blot analysis with the indicated antibodies. (f) Control and MKP-2-depleted MKK-f cells were immunostained with anti-lamin A/C antibodies to visualize microtubules and nuclear envelope, respectively. The cells were also stained with the DNA dye DAPI (40,6-diamino-2-phenylindole). Appearance of cyclin B1 on the spindle poles (arrowheads) at prometaphase was decreased in MKP-2-depleted MKK-f cells compared with control cells. Arrows indicate nuclear envelope identified by lamin A/C staining. both proliferation and survival of MKK-f cells, The immunohistochemical studies revealed that both although MKP-2 depletion in MKK-f cells did not MKP-2 and cyclin B1 are highly expressed in human affect their apoptosis. MTCs. In contrast, MKP-2 expression was low or The downregulation of cyclin B1 by the depletion undetectable in normal thyroid follicular and parafolli- of MKP-2 occurs at the transcriptional level and cular C cells. These findings suggest an in vivo role of not through the processes of translation or ubiquitin- MKP-2 in MTC development. Thus, its downregulation mediated proteolysis (Figure 5d). A recent study might become a strategy for antitumor intervention for has revealed that the activity of cyclin B1 promoter MEN2 patients. and its protein levels are upregulated by oncogenic Ras (Santana et al., 2002). Ras is also a central mediator of oncogenic signals from RET-MEN2A and RET-MEN2B (Takahashi, 2001; Asai et al., Materials and methods 2006), raising the possibility that MKP-2 may RET-MEN2A transgenic mice and cDNA microarray analyses be involved in Ras-mediated regulation of cyclin The generation of RET-MEN2A transgenic mice has been B1 expression. Much work is required to elucidate described in detail (Kawai et al., 2000). Total RNA was the mechanisms of how MKP-2 regulates cyclin B1 isolated from freshly dissected salivary, thyroid and mammary expression. tumors from RET-MEN2A transgenic mice and normal

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5692

MKP-2 cyclin B1 MEN2A MTC MEN2A normal thyroid tissue

MEN2A case 1

DAPI MKP-2 DAPI MKP-2

MEN2A case 2

Calcitonin Merged Calcitonin Merged

Normal thyroid tissue Normal thyroid tissue

MKP-2

DAPI MKP-2

cyclin B1

Calcitonin Merged

Figure 6 Expression of mitogen-activated protein kinase phosphatase (MKP)-2 and cyclin B1 in human MTC tissues from multiple endocrine neoplasia 2A (MEN2A). (a) Sections of human MTCs derived from two MEN2A patients (cases 1 and 2) were stained with anti-MKP-2 (left panel) and anti-cyclin B1 (right panel) antibodies. Brown is indicative of antibody staining. (b, c), Sections of MTC (b) and normal thyroid (c) tissues from the MEN2A patients were doubly stained with anti-MKP-2 (green) and anti-calcitonin (red) antibodies, followed by Alexa-conjugated secondary antibodies. Fluorescence was examined using a confocal laser-scanning microscope. MKP-2 was expressed in calcitonin-positive MTC tumor cells (b), but not in normal epithelial cells (arrowheads shown in b, c). MKP-2 was weakly expressed in parafollicular C cells (arrows shown in c). Cell nuclei were labeled with DAPI. (d) Sections of normal thyroid tissues were stained with anti-MKP-2 (upper panel) and anti-cyclin B1 (lower panel) antibodies. (e) The sections of normal thyroid tissues were doubly stained with anti-MKP-2 (green) and anti-calcitonin (red) antibodies. MKP-2 expression was low or undetectable in normal epithelial (arrowheads) and parafollicular C (arrows) cells.

tissues from wild-type mice. The messenger RNA (mRNA) reactions were performed using the TaqMan PCR Core was examined by differential hybridization of cDNA micro- Reagents Kit and the ABI Prism 7700 Sequence Detection array slides (‘Intelligene’ Mouse CHIP Set I; Takara, Shiga, System (Applied Biosystems). Amplification of 18S rRNA was Japan) as previously described (Kawai et al., 2003). The array used as the normalization control. was scanned with a Gene Pix 4000A (Amersham, Arlington For semiquantitative analyses of MKP-2, cyclin and CDK Heights, IL, USA) and individual spots were quantified using transcripts, total RNA from MKK-f cells was isolated using the Gene Pix Pro3.0 (Amersham). The background-corrected RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA transcripts fluorescence intensity values from each experiment were log- were then generated using Superscript II (Invitrogen, San Diego, transformed (base 2). Genes were classified as upregulated in CA, USA). RT–PCR was performed with primers specific to the tumors when a comparison with normal tissue expression mouse MKP-2 (sense, 50-CGGTTCCCCCCAGCGCCACA-30; produced a ratio with an absolute value >2.00. antisense, 50-CACTGGGATGCACTTGACT-30), mouse MKP- 3 (sense, 50-GTCTACCAGGTGGACTCCCT-30; antisense, 50-AGGAAGATATATGAAAGAGAC-30), mouse MKP-4 Quantitative and semiquantitative RT–PCR (sense, 50-ACGATGCCTACGACTTGGTC-30;antisense,50-TCA Total RNA was isolated from carcinoma samples and normal AAGACCCCATCACTGGT-30), mouse MKP-1 (sense, 50- tissues, and cDNA was synthesized following standard CCTGAGCTGTGCAGCAAA-30; antisense, 50-CTCCACAG procedures. Primers and probes for each cDNA were designed GGATCCTCTT-30), mouse MKP-5 (sense, 50-AAATTCGTC using Primer Express 1.5 software (Applied Biosystems, Foster AAAGGCAAACG-30; antisense, 50-CTTCCCTCCTTTTCG City, CA, USA). All of the real-time quantitative RT–PCR TCTCC-30), mouse MKP-6 (sense, 50-CTGGGTGTTCGGGT

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5693 TTAAGA-30;antisense,50-GAGCTCCTACTGCACCTGCT-30) silenced mouse MKP-2 expression was 50-CAATCAAGCTT and mouse PAC1 (sense, 50-TTGCCCTACCTGTACTTGGG-30; GAAAGTTA-30 (sense sequence). MKK-f cells were trans- antisense, 50-GTCTCCAGCTGTAGCAGCTG-30). fected with either the MKP-2-specific siRNA or 21-nt control siRNA (Qiagen) using Lipofectamine 2000 according to the Cell culture manufacturer’s protocol. The MKK-f cell line was established from a mammary tumor For shRNA-mediated knock down of mouse MKP-2, a set developed in a RET-MEN2A transgenic mouse (Kawai et al., of single-stranded oligonucleotides encoding the MKP-2 target 2003). MKK-f cells were maintained at 37 1C in a humidified shRNA and its complement was synthesized as follows (only the sense sequence is shown): 50-CCAAGAAATTCTTGTTA atmosphere of 5% CO2 and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% fetal bovine CA-30 (nt 2347–2365 of mouse MKP-2 cDNA). The oligo- serum (FBS). HEK293, COS7 and MEF3T3 Tet-off cells were nucleotide pair was annealed and inserted into the pNAMA grown in DMEM supplemented with 8% calf serum. retroviral shRNA expression vector (Shirane et al., 2004). GP293 packaging cells were transfected with 24 mg of control Antibodies or MKP-2 shRNA-containing pNAMA vectors, 2 mgof Mouse anti-MKP-2 monoclonal antibody was purchased from pVSVG and 60 ml of Lipofectamine 2000 reagent in 100-mm BD Transduction Laboratories (Lexington, KY, USA). Other cell culture dishes in Opti-MEM medium (Invitrogen) without antibodies used in this study include anti-phospho JNK FBS and antibiotics. The medium was replaced 24 h later, and retroviral supernatants were harvested 48 h post-transfection. monoclonal antibody, anti-phospho p42/44 MAPK polyclonal 5 antibody, anti-phospho p38 MAPK polyclonal antibody, anti- MKK-f cells (1 Â 10 ) were mixed with 3 ml of virus-containing phospho cdc2 (p-cdc2) polyclonal antibody, anti-cyclin B1 supernatant and seeded in 100-mm cell culture dishes. The monoclonal antibody (Santa Cruz Biotechnology Inc., Santa supernatant was replaced after a 24-h incubation. Clones in Cruz, CA, USA), anti-cyclin B1 polyclonal antibody (Cell which the expression of MKP-2 was effectively suppressed Signaling Technology, Beverly, MA, USA), anti-a-tubulin were selected and used for further study. monoclonal antibody (Sigma, St Louis, MO, USA), anti-lamin A/C monoclonal antibody (Santa Cruz Biotechnology Inc.) and Immunohistochemistry anti-calcitonin polyclonal antibody (Dako, Hamburg, Germany). Human MTC tissues were obtained from eight patients who underwent operations at Nagoya University Hospital. Sections Plasmid construction were incubated with the anti-MKP-2 and anti-cyclin B1 Active mutants of MAPKK (pSRa-DSESE-MAPKK) and antibodies overnight at 4 1C. After washing with PBS, they MKK6 (pcDNA3.1-MKK6-EE) were the generous gift from were treated with the secondary antibody, dextran peroxidase- Dr YGotoh (Tokyo University, Tokyo, Japan). Human full- conjugated goat anti-rabbit antibody (Envision System; Dako), length MKP-2 cDNA was isolated by PCR from a cDNA and diaminobenzidine was used to visualize immune complexes library prepared using RNA of TGW neuroblastoma cells. at room temperature. For immunofluorescent staining, sections A catalytically inactive mutant of MKP-2 (C280S) (Cadalbert were stained with anti-MKP-2 and anti-calcitonin antibodies, et al., 2005) was generated using the QuikChange Site-Directed followed by Alexa488 or 594-conjugated secondary antibodies. Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to Fluorescence was examined using a confocal laser-scanning the manufacturer’s protocol. The cDNAs were subcloned into microscope (Fluoview FV500; Olympus, Tokyo, Japan). the pEF1-myc vector (Invitrogen) and used for experiments. Western blot analyses Tet-off system Cells were lysed in SDS sample buffer (20 mM Tris-HCl, pH MEF3T3 Tet-off cells (Clontech, Palo Alto, CA, USA) were 6.8; 2 mM EDTA; 2% SDS; 10% sucrose; 20 mg/ml bromo- grown in DMEM supplemented with 10% Tet-off system- phenol blue and 80 mM dithiothreitol). The lysates were approved FBS (Clontech) containing 50 IU/ml penicillin, ultrasonicated, boiled for 4 min and subjected to SDS- 50 mg/ml streptomycin, 2 mmol/l L-glutamine and 100 mg/ml polyacrylamide gel electrophoresis. Proteins were transferred G418 (Roche Diagnostics, Mannheim, Germany), and main- to polyvinylidene difluoride membranes (Millipore, Bedford, tained at 37 1C under a 5% CO2 atmosphere. The MEF3T3 MA, USA). The membranes were blocked in 5% albumin in Tet-off cells grown in 60-mm tissue culture dishes were co- PBS containing 0.05% Tween-20 and probed with the primary transfected with 10 mg of pTRE/RET-MEN2A or pTRE/RET- antibodies. After washing, they were incubated with the MEN2B plasmid and 0.5 mg of pTK-Hygr plasmid (Clontech) secondary antibodies for 1 h before specific binding using Lipofectamine 2000 (Invitrogen) as described previously was detected by the Enhanced Chemiluminescence System (Watanabe et al., 2002). The cells were detached with trypsin 3 (Amersham). days after transfection and plated in the presence of doxycycline hydrochloride (0.01–1 mg/ml) and hygromycin B (200 mg/ml).The resulting hygromycin-resistant colonies were Cell proliferation assays 3 expanded and maintained in media supplemented with 100 mg/ MKK-f cells were cultured overnight in triplicate (10 cells per ml of G418, 100 mg/ml of hygromycin and 200 ng/ml of well in 96-well plates) in DMEM containing 8% FBS. Cell doxycycline. Expression of the RET-MEN2A and RET- proliferation was measured using WST-1 assays (Roche MEN2B proteins was induced by culturing the cells in the Applied Science, Indianapolis, IN, USA). The WST-1 reagent absence of doxycycline for 24 or 48 h. (10 ml) was added to 100 ml of cell suspension and incubated for 4 h. The enzyme-linked immunosorbent assay reader was set at a wavelength of 450 nm with a reference wavelength of 620 nm. RNA interference Data are presented as means±s.d. for each triplicate sample. The siRNA-mediated knock down of MKP-2 protein expres- sion was performed using a previously described method (Enomoto et al., 2005; Uchida et al., 2006). The 21-nt synthetic Flow cytometry duplexes were purchased from B-Bridge International Inc. Cell cycle profiles were analysed by staining intracellular DNA (San Jose, CA, USA). The targeted sequence that effectively with propidium iodide, followed by flow cytometry using a

Oncogene Roles of MKP-2 in RET-MEN2A-induced tumorigenesis T Hasegawa et al 5694 FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ, USA) Acknowledgements (Fukuda et al., 2005). We thank K Hirose (Nagoya University) for providing the Tumor growth assay in nude mice pNAMA shRNA expression vector, S Nakamura (Nagoya Tumor cells (1 Â 106) were subcutaneously injected into University Hospital) for providing sporadic and MEN2A 7-week-old female nude mice. Over a period of 4 weeks, nude MTC specimens and C Suzuki (Nagoya University) and mice were kept in sterilized, filtered cages in a 12-h light–dark T Urano (Shimane University) for helpful discussion. We also cycle under standardized environmental conditions through- thank YGotoh (Tokyo University) for providing active out the experiments. The Animal Care and Use Committee of MAPKK (pSRa-DSESE-MAPKK) and MKK6 Nagoya University Graduate School of Medicine approved (pcDNA3.1( þ )-MKK6-EE) plasmids. This study was sup- this study. ported by Grants-in-Aid for The 21st Century Center of Excellence (COE) Research, Scientific Research (A) and Data analysis Scientific Research on Priority Area ‘Cancer’ commissioned Data are presented as means±s.d. Statistical significance was by the Ministry of Education, Culture, Sports, Science and evaluated by Student’s t-test. Technology (MEXT) of Japan (to MT).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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