dusp6/mkp3 is over-expressed in papillary and poorly differentiated ...

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DUSP6/MKP3 IS OVER-EXPRESSED IN PAPILLARY AND POORLY

DIFFERENTIATED THYROID CARCINOMA AND CONTRIBUTES TO

NEOPLASTIC PROPERTIES OF THYROID CANCER CELLS

Debora Degl’Innocenti1*

, Paola Romeo1*

, Eva Tarantino2, Marialuisa Sensi

3, Giuliana Cassinelli

4,

Veronica Catalano1, Cinzia Lanzi

4, Federica Perrone

2, Silvana Pilotti

2, Ettore Seregni

5, Marco A.

Pierotti6, Angela Greco

1, and Maria Grazia Borrello

1

1 Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine,

Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;

2 Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;

3a Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan,

Italy;

3b Functional Genomics Core Facility, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;

4 Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;

5 Nuclear Medicine Division, Department of Diagnostic Imaging and Radiotherapy, Fondazione

IRCCS Istituto Nazionale dei Tumori, Milan, Italy;

6 Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

D. Degl’Innocenti and P. Romeo contributed equally to this work

Corresponding author: Dr Maria Grazia Borrello

Experimental Oncology and Molecular Medicine,

Molecular Mechanisms Unit

Fondazione IRCCS Istituto Nazionale dei Tumori,

Via GA. Amadeo, 42, Milan 20133, Italy

Email: [email protected]

Tel: 0039-02-2390-3223 Fax: 0039-02-2390-3073

Short title: DUSP6 over-expression in thyroid carcinoma

Keywords: thyroid carcinoma, dual-specificity phosphatase, DUSP6/MKP3, ERK1/2, Papillary

Thyroid Carcinoma, DUSPs

Word count: 4912

of 38 Accepted Preprint first posted on 6 November 2012 as Manuscript ERC-12-0078

Copyright © 2012 by the Society for Endocrinology.

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ABSTRACT

Thyroid carcinomas derived from follicular cells comprise papillary (PTC), follicular (FTC), poorly-

differentiated (PDTC), and undifferentiated anaplastic (ATC) carcinomas. PTC, the most frequent

thyroid carcinoma histotype, is associated with gene rearrangements that generate RET/PTC and TRK

oncogenes and with BRAF-V600E and RAS gene mutations. These last two genetic lesions are also

present in a fraction of PDTCs. The ERK1/2 pathway, downstream of the known oncogenes activated

in PTC, has a central role in thyroid carcinogenesis. In this study, we demonstrate that the BRAF-

V600E, RET/PTC, and TRK oncogenes upregulate the ERK1/2 pathway’s attenuator cytoplasmic dual-

phase phosphatase DUSP6/MKP3 in thyroid cells. We also show DUSP6 overexpression at the mRNA

and protein levels in all of the analysed PTC cell lines. Furthermore, DUSP6 mRNA was significantly

higher in PTC and PDTC in comparison to normal thyroid tissues both in expression profile datasets

and in patients’ surgical samples analysed by real-time RT-PCR. Immunohistochemical and Western

blot analyses showed that DUSP6 was also overexpressed at the protein level in most PTC and PDTC

surgical samples tested, but not in ATC, and revealed a positive correlation trend with ERK1/2

pathway activation. Finally, DUSP6 silencing reduced the neoplastic properties of four PTC cell lines,

thus suggesting that DUSP6 may have a pro-tumourigenic role in thyroid carcinogenesis.

Non-standard abbreviations:

ATC anaplastic thyroid carcinoma

DUSPs dual-specificity phosphatases

FFPE formalin fixed paraffin embedded

FTC follicular thyroid carcinoma

IHC immunohistochemical analyses

INT Fondazione IRCCS Istituto Nazionale

dei Tumori

MKPs MAP-kinase phosphatase

PDTC poorly-differentiated thyroid carcinoma

PTC papillary thyroid carcinoma

pTNM pathological tumor-node-metastasis

staging

SRB sulforhodamine B

TC thyroid cancer

TTF1 thyroid transcription factor 1

WB western blot

WDTC well-differentiated thyroid cancer

EMT epithelial to mesenchymal transition

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INTRODUCTION 1

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Thyroid cancer (TC) is the most common endocrine malignancy, with an increasing incidence over the 3

last few decades (Davies et al., 2006). Most TC, including well-differentiated (WDTC), poorly-4

differentiated (PDTC), and undifferentiated anaplastic (ATC) carcinomas, originates from thyrocytes. 5

Papillary thyroid carcinoma (PTC), a WDTC histotype, is the most prevalent thyroid malignancy. It is 6

usually associated with a good prognosis and therapeutic response; nevertheless, approximately 10% of 7

patients present with recurrences and distant metastases. Four different alternative genetic lesions have 8

been identified as driving oncogenic alterations in approximately 70% of PTCs, including RET or TRK 9

rearrangements and BRAF or RAS mutations. All of these PTC-associated genetic lesions constitutively 10

activate the ERK1/2 pathway (Greco et al., 2009). PDTC, recently recognised as an independent 11

histotype, presents morphological and behavioural characteristics intermediate between those of 12

WDTC and ATC. Both PDTC and ATC can arise de novo or can evolve from pre-existing WDTC, 13

particularly from PTC. Accordingly, WDTC-associated gene mutations are also found in small 14

fractions of PDTC (BRAF and RAS mutations) and ATC (BRAF mutation) (Santoro et al., 2002; 15

Santarpia et al., 2008). 16

The MAP-kinases/ERK1/2 pathway plays well-recognised roles in cell proliferation, differentiation, 17

survival, and motility. The activity of ERK1/2 is tightly regulated by many broad- and narrow-18

specificity phosphatases in physiological and pathological contexts (Chambard et al., 2007). MAP-19

kinase phosphatase enzymes (MKPs), which belong to the family of dual-specificity phosphatases 20

(DUSPs), inactivate different MAPK proteins, including ERK1/2. Among these, DUSP6/MKP3 21

cytoplasmic phosphatase displays a high specificity for ERK1/2 (Groom et al., 1996; Muda et al., 1998; 22

Camps et al., 1998; Fjeld et al., 2000; Arkell et al., 2008). Recently, p38 and FOXO1 have been 23

suggested as additional DUSP6 targets (Wu et al., 2010; Zhang et al., 2011). 24

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DUSP6 is an evolutionarily conserved, strictly regulated gene required during development, whose 25

product is subject to regulation at multiple levels, including mRNA transcription and stability, rate of 26

translation, protein stability and enzymatic activity (Bermudez et al., 2010). The MEK-ERK1/2 27

pathway appears to be a major regulator of DUSP6, as activated ERKs induce DUSP6 mRNA 28

transcription, and MEK-dependent phosphorylation of the DUSP6 protein is followed by proteasomal 29

degradation (Bermudez et al., 2011). 30

The role of DUSP6 in neoplastic transformation is poorly defined, and either up- or downregulation of 31

this phosphatase has been reported in different tumours. DUSP6 expression is low in invasive 32

pancreatic adenocarcinoma, lung, oesophageal and nasopharyngeal carcinomas (Furukawa et al., 2003; 33

Furukawa et al., 2005; Okudela et al., 2009). By contrast, DUSP6 is upregulated in myeloma cell lines 34

with an active NRAS mutation, melanoma cell lines with BRAF or NRAS mutations, colon carcinoma 35

and HER2-positive breast cancers (Croonquist et al., 2003; Bloethner et al., 2005; Lucci et al., 2010; 36

Quyun et al., 2010). In addition, DUSP6 pro-survival functions have been hypothesised in HeLa 37

(MacKeigan et al., 2005) and breast cancer cells (Lonne et al., 2009), and a tumour-promoting role has 38

recently been suggested in glioblastoma cells (Messina et al., 2011). 39

As shown in the flowchart in Figure S1, we investigated DUSP6 expression in PTC and PDTC, 40

starting from the evidence that the RET/PTC1 oncogene, known to enhance ERK pathways in primary 41

human thyrocytes (Borrello et al., 2005), concomitantly upregulates certain regulators of these 42

pathways, including several DUSPs and SPRY2. We have shown that high levels of DUSP6 mRNA 43

and protein are present in all of the analysed PTC cell lines and in the majority of PTC and PDTC 44

surgical samples. Unexpectedly, high levels of the protein were associated with high ERK1/2 activation 45

in the analyzed TCs. Functional experiments of DUSP6 silencing in four PTC cell lines that 46

overexpress the phosphatase unveiled a pro-tumorigenic role for DUSP6. 47

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MATERIALS and METHODS 49

50

Antibodies and reagents 51

The following mouse monoclonal Abs were used in blotting experiments: anti-DUSP6 from Abcam 52

(Cambridge, UK); anti-MEK1/2 from Cell Signaling Technology (Beverly, MA, USA); anti-MAP 53

kinase activated (pERK1/2) and anti-vinculin from Sigma-Aldrich (Steinheim, Germany). The 54

following rabbit monoclonal Ab was used in blotting experiments: anti-phospho-Akt (Ser473) from 55

Cell Signaling Technology. The following rabbit polyclonal Abs were used in blotting experiments: 56

anti-RET, anti-TRK, anti-RAF-B, anti-RSK-1, p-RSK-1/2 (Thr359/Ser363) and anti-p-Shc (Tyr 57

239/240) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-MAP kinase (ERK1/2) from 58

Sigma-Aldrich; anti-phospho-MEK1/2 (Ser217/221), anti-Akt, anti-PARP and anti-cleaved PARP 59

(Asp214) from Cell Signaling Technology; anti-NBS1 from Novus Biologicals (Littleton, CO, USA) 60

and anti-Shc from Upstate (Lake Placid, NY, USA). 61

EGF was from Sigma-Aldrich. The MEK inhibitor UO126 was from Promega (Madison, WI, USA). 62

Human BRAF-V600E cDNA cloned in the pMCEF vector, kindly donated by Dr. R. Marais 63

(Wellbrock et al., 2004), was subcloned into the pRC-CMV vector. Human RET/PTC1 and TRK-T3 64

cDNAs were cloned in the pRC-CMV vector (Roccato et al., 2002). 65

66

Tumour samples 67

Thyroid samples were collected at the Department of Pathology at Fondazione IRCCS Istituto 68

Nazionale dei Tumori (INT), Milano, Italy. All patients signed an informed consent for the 69

experimental use of their tissue samples in this study, which was approved by the Independent Ethical 70

Committee of INT. 71

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The TC were classified according to WHO Classification (Delellis et al., 2004), and the extent of 72

disease was determined according to the pathological tumour-node-metastasis (pTNM) staging system 73

(Sobin et al., 2009). Genetic lesions were characterised as previously described (Frattini et al., 2004). 74

The non-neoplastic thyroid tissues were from patients with pathologies other than TC. Twenty frozen 75

thyroid samples (including five non-neoplastic and 15 TC) were selected for real-time RT-PCR 76

analyses (Table S2 and Figure 4). An additional fifteen formalin-fixed, paraffin-embedded (FFPE) 77

thyroid samples (including two non-neoplastic, three PTCs, eight PDTCs and two ATCs) were 78

investigated by immunohistochemical (IHC) analyses. Among these, five pairs of matched frozen 79

tissues (including two non-neoplastic and three PDTCs) were analysed by Western Blot (WB). 80

81

RNA extraction and real-time RT-PCR analysis 82

Total RNA from thyrocytes was extracted using NucleoSpin®

RNA II (Macherey-Nagel, Düren 83

Germany), following the manufacturer’s protocols. Total RNA from tissue specimens was extracted as 84

previously described (Frattini et al., 2004). Total RNA was reverse-transcribed using the High-85

Capacity cDNA Archive Kit (Applied Biosystems, Foster City, California, USA). For each sample, 20 86

ng of template was amplified in PCR reactions performed in triplicate on an ABI PRISM 7900 using 87

the TaqMan®

Gene Expression Assay (Applied Biosystems). DUSP4, DUSP6, SPRY2, PLAU and 88

CSF2 were tested. PGK1 was used as a housekeeping gene. Data analyses were performed with the 89

SDS (Sequence Detection System) 2.4 and the RQ Manager 1.2.1 programs, using the 2−∆∆Ct

method 90

with a relative quantification RQmin/RQmax confidence level set at 95%. The error bars display the 91

calculated maximum (RQmax) and minimum (RQmin) expression levels that represent SE of the mean 92

expression level (RQ value). The upper and lower limits define the region of expression within which 93

the true expression level is likely to occur. 94

95

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Microarray data analysis 96

The expression of ERK pathway attenuators DUSP5 and DUSP6 and that of DUSP4, DUSP10, 97

SPRED2 and SPRY2, when available, was examined in two microarray datasets from thyroid tissues 98

that comprised 69 PTCs and 13 non-neoplastic thyroids. One dataset, generated in our laboratory using 99

cDNA microarray chips, contains the expression profile data of 9 non-neoplastic thyroids and 34 PTC 100

collected at the Department of Pathology of our Institute. The PTC collection includes 24 classical 101

types and ten Tall Cell variants; 11 samples carry the BRAF-V600E mutation, seven samples carry 102

RET/PTC rearrangements, two samples carry TRK rearrangements, and for the remaining five samples, 103

none of the above genetic lesions was detected. The details of gene expression analysis have been 104

previously reported (Frattini et al., 2004). The other dataset examined was extracted from the NCBI 105

Gene Expression Omnibus (GEO) database under series number GSE27155 (Giordano et al., 2005). It 106

contained the expression profile performed using oligonucleotide DNA microarrays (U133A 107

GeneChip, Affymetrix, Santa Clara, CA, USA) with thyroid samples, including four normal thyroid 108

tissues and 35 PTC corresponding to classical (25) and Tall Cell (10) types. Among PTC samples, 26 109

carried the BRAF-V600E mutation and eight had RET/PTC rearrangements. The log intensity value of 110

probesets corresponding to DUSP4 (204014_at, 204015_s_at), DUSP5 (209457_at), DUSP6 111

(208891_at, 208892_s_at 208893_s_at), DUSP10 (215501_s_at, 221563_at), SPRY2 (204011_at) and 112

SPRED2 (12458_at, 212466_at) were considered. Because the multiple DUSP4, DUSP6, DUSP10 and 113

SPRED2 probesets displayed an identical trend in transcript level changes, the average log intensity 114

levels of the different probesets for the same gene are reported. 115

116

Cell culture, transfections and RNA interference 117

Primary thyrocyte cultures were established from non-neoplastic thyroid samples from patients 118

undergoing surgery at INT and were maintained in a nutrient mixture consisting of Ham’s F12 medium 119

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(custom-made by Invitrogen, Paisley, UK) containing 5% calf serum and bovine hypothalamus and 120

pituitary extracts, as previously described (Curcio et al., 1994). Primary thyrocytes expressing the 121

RET/PTC1 oncogene, obtained by infection with RET/PTC1 retroviral vector, have been described 122

(Borrello et al., 2005). Immortalised cell lines were maintained in media supplemented with 10% calf 123

serum. The human thyroid cell lines NIM-1, TPC1, B-CPAP, WRO, 8505C, KAT4, KAT18, BHT101 124

and HTC/C3 were grown in DMEM; N-Thy-ori3-1 were grown in RPMI-1640; K1 in DMEM: Ham’s 125

F12: MCDB; FTC133 in DMEM: Ham’s F12; and HOTHC in Ham’s F12. 126

N-thy-ori3-1 cells were transiently transfected using the Cell Line Nucleofector Kit V (Lonza, Basel, 127

Switzerland), program X-005, according to manufacturer’s protocols. Knockdown of DUSP6 protein in 128

TPC1, NIM-1, B-CPAP and K1 cells was performed by transfection with the ON-TARGET plus 129

SMART pool for human DUSP6 or NON-TARGET small interfering RNA control (Thermo Scientific, 130

Dharmacon Inc. Chicago, IL, USA) using siIMPORTER transfection reagent (Millipore, Billerica, MA, 131

USA), following the manufacturer’s instructions. 132

133

Western blot analysis 134

Total protein cell extracts were prepared as previously described (Degl'Innocenti et al., 2010). 135

For separation of nuclear and cytoplasmic proteins, cells were incubated in a hypotonic buffer (10 mM 136

HEPES pH 7.9, 10 mM MgCl2, 0.5% NP-40, 0.5 mM DTT) supplemented with protease and 137

phosphatase inhibitors. Subsequently, nuclei were sedimented by centrifugation and lysed through 138

sonication in a high-salt buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 0.5 mM EDTA, 1.5 mM 139

MgCl2, 25% glycerol, 0.5 mM DTT) supplemented with protease and phosphatase inhibitors. 140

141

Immunohistochemical (IHC) analysis 142

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The IHC experiments were performed with the antibodies and under the conditions shown in Table 1 143

using adequate positive and negative controls. For the 15 cases analysed through IHC, representative 144

sections were selected and immunophenotyped. 145

146

DNA isolation and sequencing 147

Genomic DNA from FFPE specimens was isolated using the Qiagen Tissue Kit (Qiagen, Chatsworth, 148

CA, USA) as previously described (Namba et al., 2003). We analysed exons 11 and 15 of BRAF 149

through DNA amplification using specific primers (Davies et al., 2002; Namba et al., 2003). The 150

primers for BRAF analysis included the exonic sequence and at least 50 nucleotides of the flanking 151

intronic sequences. Amplified products were purified with the QIAamp Purification Kit (Qiagen) and 152

then directly sequenced on an ABI PRISM 3100 automated capillary Genetic Analyzer (Applied 153

Biosystems). 154

155

Early branching morphogenesis assay 156

Morphogenic properties of thyroid cells were evaluated by testing cells’ ability to aggregate and form 157

branches in a few hours when layered on an artificial extracellular matrix (Matrigel; BD Biosciences, 158

San Jose, CA, USA), as previously described (Cassinelli et al., 2009). Seventy-two hours after 159

transfection, cells were suspended in serum-free medium and overlaid on the gelled Matrigel. After 160

incubating at 37°C for 4 hours, branches were photographed with a digital camera. Quantification of 161

branches was performed by measuring the total length of structures per field in adjacent fields (n=10). 162

The data are reported as percentages of control ± SD. 163

164

Cell proliferation assays 165

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Twenty-four hours after transfection, siRNA-transfected and untransfected control cells were seeded at 166

20,000 cells/cm2 in 96-well plates in the presence of DMEM with 10% FBS. Six hours after seeding, 167

the cells were serum-starved and exposed to solvent or drug when indicated. Cell growth was evaluated 168

by sulphorhodamine B (SRB) colorimetric assay at the indicated times, as previously described 169

(Degl'Innocenti et al., 2010). The experiments were performed in eight replicates. 170

Cell migration and invasion assays 171

Forty-eight hours after transfection, PTC cells were harvested and transferred into 24-well transwell 172

chambers (Costar, Corning, Inc., Corning, NY) in complete medium. For the migration assay, cells 173

were seeded in the upper chamber. For the invasion assay, the transwell membranes were coated with 174

Growth Factor Reduced Matrigel (12.5 µg in 60 µl/well) (BD Biosciences) and dried for one hour. 175

Cells were transferred onto the artificial basement membrane. After 24 hours of incubation at 37°C, 176

cells that invaded the Matrigel layer and/or migrated to the lower chamber were fixed in 95% ethanol, 177

stained with a solution of 0.4% SRB in 1% acetic acid, and counted under an inverted microscope. 178

Assays were performed in triplicate, cells were counted in adjacent fields (n=10), and data were 179

reported as average cell number per field ± SD. 180

181

Apoptosis analysis 182

Cells were fixed and stained with Hoechst 33341 (Sigma) as previously described (Cassinelli et al., 183

2009). Apoptosis was evaluated by counting Hoechst 33341-stained apoptotic bodies in adjacent fields 184

(n=10) and expressed as the average percentage ± SD. 185

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Statistical analyses 187

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Statistical analyses and graphs were generated using GraphPad Prism version 5.0. Comparison between 188

two groups was performed with the two-tailed Student’s t-test or the Mann-Whitney U-test, as stated in 189

the figure legends. Three or more groups were analysed with the Kruskal-Wallis test with Dunn’s 190

multiple comparison post-test. p<0.05 was considered significant. Asterisks indicate P<0.05 (*), 191

P<0.01 (**), and P<0.001 (***). 192

193

RESULTS 194

The RET/PTC1 oncogene upregulates attenuators of ERK pathways in primary human 195

thyrocytes 196

Using human primary thyrocytes exogenously expressing the RET/PTC1 oncogene as an in vitro model 197

of PTC, we have previously shown through microarray analysis (U133 GeneChips, Affymetrix) that 198

RET/PTC1 induces the expression of a large set of genes, including genes involved in inflammation 199

and tumour invasion. Their induction is strictly dependent on the presence of the RET/PTC1 major 200

docking site, Tyr451 (Borrello et al., 2005). 201

In this work, we have further analysed the previously obtained gene expression profiles of uninfected 202

human primary thyrocytes and of RET/PTC1- or RET/PTC1-Y451F-infected cells (Borrello et al., 203

2005). This novel analysis of expression profiles revealed the oncogene-induced upregulation of 204

several MAPK pathway attenuators, including SPRY2, SPRED2, DUSP4, DUSP5, DUSP6 and 205

DUSP10 (Figure 1A). 206

By real-time RT-PCR analysis, we have now validated the expression of selected genes (Figure 1B). 207

The mRNAs of DUSP4, DUSP6 and SPRY2 were found to be up to 200-fold more abundant in 208

RET/PTC1- with respect to RET/PTC1-Y451F-infected or uninfected thyrocytes. The established 209

RET/PTC’s transcriptional target PLAU and CSF2 genes have been used as controls (Borrello et al., 210

2005; Guarino et al., 2009). These findings suggest that RET/PTC1 concomitantly activates the 211

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ERK1/2 pathway and several potential regulators of this pathway, both largely dependent on the RET 212

multi-docking site. 213

214

Attenuators of ERK pathways in PTC gene datasets and in TC cell lines 215

To assess whether the ERK pathway attenuators upmodulated in vitro by RET/PTC1 could be 216

overexpressed in PTC clinical samples, we examined two microarray datasets of thyroid tissues for a 217

total of 13 non-neoplastic thyroids and 69 PTCs, including different subtypes. For both datasets, 218

information about the genetic alteration of the neoplastic samples was available. 219

In the first dataset, a cDNA array generated in our laboratory (Frattini et al., 2004), only DUSP5 and 220

DUSP6 could be investigated. All six of the MAPK feedback genes could be investigated in the second 221

dataset downloaded from GEO (series number GSE27155) (Giordano et al., 2005). These analyses 222

(Figure 2) indicate that DUSP4, DUSP5, DUSP6 and SPRED2 expression is significantly higher in 223

PTC compared to non-neoplastic thyroid, while DUSP10 and SPRY2 are expressed at similar levels. 224

With regard to PTC histotypes, no difference could be observed between PTC NOS (not otherwise 225

specified) and the more aggressive Tall Cell variant for the analysed genes (data not shown). 226

Upregulation of DUSP4, DUSP5 and DUSP6 in PTCs confirms published data from additional 227

independent microarray studies (Huang et al., 2001; Chevillard et al., 2004; Jarzab et al., 2005; Griffith 228

et al., 2006; Delys et al., 2007; Eszlinger et al., 2007; Salvatore et al., 2007; Arora et al., 2009; 229

Fontaine et al., 2009;). The expression profiles of ERK pathway attenuators indicate that DUSP4-5-6 230

overexpression is significant in PTCs irrespective of their genetic lesion (Figure S2). 231

To confirm this finding, the mRNA levels of DUSP4, DUSP6 and SPRY2 were analysed by real-time 232

RT-PCR in cell lines representative of PTC, FTC and ATC harbouring different genetic alterations 233

(Table S1), in comparison with immortalised normal thyrocytes (N-thy-ori3-1). As shown in Figure 234

S3, these three genes were expressed poorly or not at all in immortalised N-thy-ori3-1 cells. The 235

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overexpression of DUSP6 (200-600 fold), was observed in all PTC but not in FTC cell lines (panel A). 236

DUSP4 was overexpressed in almost all of the ATC and in two of four PTC cell lines (panel B). SPRY2 237

was moderately overexpressed in all of the PTC lines (panel C). Taken together, these results indicate 238

that PTC cells overexpress several members of the DUSP family compared with N-thy-ori3-1 cells. 239

DUSP6 was distinctly overexpressed in the PTC histotype, and ERK1/2 pathway-specific mechanisms 240

(Groom et al., 1996; Muda et al., 1998; Arkell et al., 2008), have been further investigated. 241

242

DUSP6 upregulation by PTC-associated oncogenes depends on ERK1/2 pathway activation 243

The effect of different PTC-related oncogenes on DUSP6 expression was investigated in N-thy-ori3-1 244

cells exogenously expressing BRAF-V600E, RET/PTC1 or TRK-T3. All PTC-associated oncogenes 245

induced DUSP6 upregulation compared to mock-transfectants (Figure 3A). Accordingly, the inhibition 246

of RET/PTC1 by the RET-targeting agent RPI-1 (Cassinelli et al., 2009) in TPC1 cell line was 247

associated with abrogation of DUSP6 expression (data not shown). The strongest DUSP6 modulation 248

was induced by BRAF-V600E, in keeping with observations of PTC cell lines that endogenously 249

express this oncogene. 250

Because we have demonstrated that the RET/PTC1 multi-docking site, responsible for MEK-ERK1/2 251

pathway activation, is necessary for DUSP6 upregulation, and the highest DUSP6 expression was 252

present in BRAF-V600E cells, we next evaluated DUSP6 expression levels in TPC1 and K1 cells 253

treated with the MEK inhibitor UO126. DUSP6 mRNA was strongly downregulated in drug-treated 254

cells compared to control cells (Figure 3B), suggesting that DUSP6 overexpression might be a 255

compensatory mechanism in response to inhibition of the ERK1/2 pathway. DUSP6 protein was also 256

downregulated in response to treatment with the MEK inhibitor in both TPC1 and K1 cells (panel B, 257

right). DUSP6-specific antibody detected two protein bands corresponding to translation products 258

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initiating at different ATG codons, and the larger protein was more greatly affected by treatment 259

(Figure 3B), as previously shown (Zhang et al., 2010). 260

DUSP6 protein was variably overexpressed in all of the analysed PTC cells (Figure 3C). Accordingly, 261

selected components of the MAP-kinase pathway were found to be more highly phosphorylated in all 262

of the analysed PTC cell lines, compared to N-thy-ori3-1. Within the ERK signalling cascade, 263

MEK1/2, the upstream activators of ERK1/2, were found to be phosphorylated in BRAF-V600E- and, 264

to a lesser extent, in RET/PTC1-expressing cells. The ERK effectors RSK1/2 were mostly activated in 265

BRAF-V600E expressing cells. Overall, the expected inverse correlation between DUSP6 expression 266

and ERK1/2 activation was not observed. Moreover, nuclear/cytoplasmic fractionation of proteins was 267

performed, and DUSP6 displayed an exclusive cytoplasmic localisation, as expected, and total and 268

phosphorylated ERK1/2 proteins were mostly cytoplasmic (Figure 3D). 269

270

DUSP6 mRNA and protein expression in human thyroid carcinoma surgical samples 271

We next analysed DUSP6 mRNA expression by real-time RT-PCR in five non-neoplastic thyroids and 272

15 PTC biopsies, including nine primary tumours from pT1 to pT4 stage, and six nodal metastasis. The 273

genetic and histological characterisation of the PTCs cases is reported in Table S2. As shown in 274

Figure 4A, DUSP6 transcripts were significantly higher in PTCs than in non-neoplastic thyroids. 275

According to our results in TC patient datasets and cell lines (Figures 2 and S2), the overexpression of 276

DUSP6 in PTCs cases was independent of the harboured genetic lesion (Figure 4B). The same PTC 277

samples were grouped into primary tumours, divided into stage 1–2 and stage 3–4, and nodal 278

metastases (panels C and D). DUSP6 transcript levels remained significantly upregulated compared to 279

non-neoplastic thyroids for the pT3-T4 and nodal subclasses and trended upwards with tumour stage 280

and in nodal metastases vs. primary tumours. 281

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To further investigate the expression of DUSP6 in TC, we performed IHC analysis for the detection of 282

DUSP6 protein and ERK phosphorylation in a series of TC samples characterised by increasing 283

aggressiveness. The cases analysed included two non-neoplastic samples, three PTCs (one NOS, one 284

tall cell and one follicular variant), eight PDTCs, and two ATCs. Of note, three PDTCs and one ATC 285

retained a papillary component, suggesting that they were derived from pre-existing PTCs. The results 286

are reported in Table 2. Very low expression of DUSP6 was found in normal tissues and in ATCs, 287

whereas DUSP6 was upregulated in the three PTCs and in 7/8 PDTCs, despite its heterogeneous levels. 288

Figure 5 shows representative cases including one non-neoplastic thyroid, three PDCTs (panel A), and 289

one PTC/PDTC case showing two histologically distinct components (panel B). IHC analysis showed 290

that DUSP6 was highly expressed in cases #10 and #13 compared to non-neoplastic thyroid and was 291

found in the cytoplasm, as expected. ERK1/2 total proteins were expressed at similar levels in non-292

neoplastic and tumour tissues and displayed both nuclear and cytoplasmic localisation. Phosphorylated-293

ERK1/2 proteins were absent in non-neoplastic thyroids, in 1/3 PDTCs (#9, displaying low DUSP6) 294

and in 2/2 ATC. By contrast, pERK1/2 were easily detected in the other ten PDTC and PTCs samples 295

(cases #10 #13 are shown). The marker of thyroid differentiation TTF1 (thyroid transcription factor 1) 296

was analysed as a control (Bejarano et al., 2000) (Table 2, Figure 5A). Interestingly, case #7 presented 297

two histologically distinct tumour components: PDTC and PTC tall cell. BRAF gene sequence analysis 298

from the dissected FFPE sample revealed the presence of BRAF-wt in PDTC and BRAF-V600E in the 299

PTC area (data not shown). In the latter, DUSP6 was strongly upregulated, and ERK1/2 was markedly 300

activated compared to the BRAF-wt area (Figure 5B). 301

Furthermore, to confirm the IHC results and to extend the analysis to other components of the ERK 302

pathway, five thyroid samples for which matched frozen tissue was available were subjected to WB 303

with antisera to DUSP6 and to total and phosphorylated ERK1/2 and MEK proteins (Figure S4). 304

DUSP6 expression levels and ERK phosphorylation in thyroid samples by WB analysis correlated with 305

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those obtained by IHC, as shown in Figure 5A. MEK1/2 were found activated in the PDTC sample 306

(#10) displaying the highest ERK1/2 phosphorylation level. 307

Altogether, our analyses of surgical specimens unveiled DUSP6 protein overexpression in most 308

PTC/PDTC cases and a positive correlation with ERK1/2 pathway activation. 309

310

DUSP6 biological effects 311

The functional role of DUSP6 in PTC was investigated through its silencing in the PTC cell lines 312

TPC1, NIM-1, K1 and B-CPAP. As described in Table S1, TPC1 carry the RET/PTC1 and the other 313

cell lines the BRAF-V600 oncogene. We have previously shown that TPC1 cells display morphogenic 314

properties that are abrogated by treatment with the RET inhibitor RPI-1 (Cassinelli et al., 2009). TPC1 315

cells transiently transfected with DUSP6 small interfering RNAs (siDUSP6), which drastically reduced 316

the DUSP6 expression level (Figure 6A), showed a significant reduction in morphogenic capability 317

compared to non-targeting siRNA-transfected cells. Biochemical analyses showed, subsequent to 318

DUSP6 silencing, a marked reduction of ERK1/2 phosphorylation (50-70% in repeated experiments). 319

PARP analysis (panel A) and apoptotic bodies count (data not shown) suggest that siDUSP6 does not 320

cause apoptosis. The proliferation rate of siDUSP6-transfected TPC1 cells was lowered only five days 321

after transfection (Figure 6B and data not shown). In a separate series of experiments (Figure 6C), 322

DUSP6 silencing significantly reduced the ability of TPC1 cells to migrate into the lower chamber of a 323

transwell and to invade the Matrigel layer. Parallel biochemical analysis showed that DUSP6 silencing, 324

in addition to lowering pERK levels, consistently lowers pMEK and, to a lesser extent, p52 and p66 325

SHC protein phosphorylation. Interestingly, siDUSP6 slightly decreased RET/PTC1 phosphorylation 326

without affecting the activation of the RTK most active in TPC1 cells, including HGFR, EGFR and 327

AXL (Figure S5) 328

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NIM-1 cells, previously demonstrated to depend on RAF/MEK/ERK activation for proliferation 329

(Degl'Innocenti et al., 2010), were similarly transfected with siDUSP6 or non-targeting siRNA (Figure 330

7). As a further control, untransfected cells were treated with UO126. As expected, the MEK inhibitor 331

significantly reduced NIM-1 cell growth (panel A, left) and abolished ERK1/2 phosphorylation and 332

concomitantly activated Akt (panel A, right and (Degl'Innocenti et al., 2010)). DUSP6 silencing 333

significantly reduced NIM-1 cell growth, as did UO126. Biochemical analysis showed that DUSP6 334

silencing was associated with slight but reproducible lowering of ERK1/2- and Akt-phosphorylation. 335

Cleaved PARP was clearly induced by DUSP6 silencing in NIM-1 in contrast with UO126 treatment. 336

Apoptosis induction by siDUSP6 was also confirmed by immunofluorescent detection of apoptotic 337

cells (panel B). 338

Because untreated NIM-1 cells have invasive but not morphogenic capabilities (data not shown), the 339

effects of siDUSP6 on NIM1 cells were assessed through migration and invasion assays (panel C). 340

NIM-1 cells transfected with siDUSP6 showed significantly reduced ability to migrate and to invade 341

the Matrigel layer compared to non-targeting siRNA-transfected cells. Similarly, DUSP6 silencing 342

significantly reduced the invasive behaviour of the PTC cell lines K1 and B-CPAP (Figure S6). 343

Overall, our functional experiments in four PTC cell lines (summarised in Figure S1) suggest that 344

DUSP6 silencing counteracts malignant PTC cells phenotypes. 345

346

DISCUSSION 347

We have shown that PTC cell lines and the majority of PTC and PDTC specimens overexpress 348

DUSP6/MKP3. Accordingly, DUSP6 displays tumour-promoting effects in TC cell lines. 349

It is known that the ERK1/2 pathway is essential for thyroid carcinogenesis (Greco et al., 2009) and 350

that DUSP6 mediates one of the feedbacks to this pathway. We have demonstrated that BRAF, 351

RET/PTC and TRK oncogenes activated in TC are able to upregulate DUSP6 expression, thus 352

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activating both the ERK1/2 pathway and its negative feedback mechanisms. Consistently, we found 353

that other ERK signalling regulators are upregulated by RET/PTC1 and are overexpressed in PTC 354

datasets. However, the ERK1/2-DUSP6 interplay is complex, as active ERK1/2 upregulates DUSP6 355

mRNA, but by favouring protein degradation, downregulates DUSP6 protein (Bermudez et al., 2011). 356

Analyses of public gene expression profiles and of our surgical samples concordantly suggest that most 357

PTC overexpress DUSP6 mRNA. Through IHC and biochemical analyses, we have demonstrated that 358

DUSP6 protein was overexpressed in TC surgical samples compared to non-neoplastic thyroids and 359

thyrocytes surrounding the tumour. This was a novel finding because DUSP6 has mainly been 360

investigated at the RNA level in cancers. 361

Because it has been suggested that DUSP6 acts as a tumour suppressor gene in several carcinomas, it 362

might also be hypothesised to have a similar function in TC progression. On the contrary, we have 363

found DUSP6 overexpression even in more aggressive PTC variants and in PDTC, a histotype with 364

features intermediate between WDTC and ATC. The only PDTC that did not overexpress DUSP6 365

showed basal levels of ERK1/2 pathway activation. The same was true for the two analysed ATCs: 366

neither overexpress DUSP6, in accord with literature on DUSP6 mRNA (Salvatore et al., 2007), and 367

both show basal levels of pERK1/2 (data not shown). A positive correlation trend was found between 368

DUSP6 expression and the activation of the ERK1/2 pathway components MEK, ERK1/2 and RSK. 369

Thus, DUSP6 overexpression seems to be a read-out of ERK1/2 pathway activation instead of being its 370

negative feedback. This was corroborated by a reported specific case displaying high pERK and 371

DUSP6 levels in a BRAF-V600E-positive tumour area and low pERK and DUSP6 in a BRAF-wt 372

tumour area. 373

Upregulation of DUSP6 has been reported in tumours of different histotypes, (e.g. Quyun et al., 2010). 374

In addition, a tumour-promoting role for DUSP6 has recently been suggested in glioblastoma cells 375

(Messina et al., 2011). 376

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We performed functional experiments in four PTC cell lines (TPC1, NIM-1, K1, and BCPAP). DUSP6 377

silencing in TPC1 cells resulted in a reduction of branched morphogenesis, consistent with inhibition of 378

the EMT. This finding is in agreement with the reported identification of DUSP6 as one of the GDNF-379

induced genes regulated by the RET proto-oncogene during ureteric bud branching morphogenesis (Lu 380

et al., 2009). Of note, DUSP6 was necessary but not sufficient to induce branching morphogenesis 381

because the other three PTC cell lines do not show this ability. Interestingly, DUSP6 silencing 382

significantly reduced the invasive ability of all four PTC cell lines. In addition, NIM-1 cell proliferation 383

was reduced and apoptosis was enhanced. Furthermore, in both NIM-1 and TPC1 cells, the steady state 384

level of ERK1/2 was not enhanced by DUSP6 silencing, as might be expected by lowering a negative 385

feedback regulator. This apparent discrepancy might be the result of the complex network involving 386

forward and feedback regulators of RTK and ERK1/2 pathways (Wortzel et al., 2011). How the 387

shutdown of DUSP6 may lower pERK1/2 and the thyroid cell lines’ invasive and migratory abilities 388

remains to be elucidated, especially considering the hundreds of protein substrates of these kinases. Of 389

note, our results suggest a possible backward effect of DUSP6 on RET/PTC1 protein activation in 390

TPC1 cells. Furthermore, in NIM-1 cells, we showed that DUSP6 silencing, by contrast with ERK1/2 391

chemical inhibition, enhances apoptosis and lowers pAKT, thus confirming the known interplay 392

between ERK and AKT pathways in the thyroid (Miller et al., 2009). Although we cannot exclude the 393

role of additional DUSP6 targets, the phosphorylation of p38, recently indicated as a novel DUSP6 394

target, is not enhanced through DUSP6 silencing (data not shown). 395

Although further studies are needed, our work clearly points to DUSP6 overexpression as a possible 396

player in thyroid malignancy. High DUSP6 expression levels in PTC were confirmed by Lee et al. in 397

work published during the review process of our manuscript (Lee et al., 2012). 398

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Overall, our work suggests that dissecting the role of ERK1/2 pathway components may allow a better 399

understanding of the complex network involved in thyroid carcinogenesis, possibly providing useful 400

information to design appropriate targeted therapies. 401

402

Declaration of interest 403

The authors declare that there is no conflict of interest that could be perceived as prejudicing the 404

impartiality of the research reported. 405

Funding 406

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) and 407

Institutional Strategic Projects ‘contribution 5 per mille’ Fondazione IRCCS Istituto Nazionale Tumori. 408

Author contributions 409

MGB and DD designed the study with the collaboration of SP, AG, ES, PP, and MAP. PR, GC, VC 410

and CL conceived, performed and analyzed data of the in vitro and functional experiments. DD 411

conceived, performed and analyzed data of RealTime experiments. MS performed dataset analysis. 412

ET and FP performed histopathological analysis evaluated by SP. ES collected clinical data. All 413

authors were involved in writing the paper, especially MGB, DD, SP, PR, MS, AG, CL and MAP. 414

Acknowledgements 415

We wish to thank Dr Elena Tamborini for helpful methodological advice and discussion, Miss Maria 416

Grazia Rizzetti, Mrs Enrica Favini and Mrs Laura Dal Bo for technical assistance, Dr Richard Marais 417

for kindly donating BRAF-V600E expressing plasmid, and Silvia Grassi for secretarial assistance. 418

419

420

421

422

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577

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Figure Legends 579

Figure 1 The RET/PTC1 oncogene elicits upregulation of ERKs pathway attenuators. (A) 580

Expression plot of MAPK pathway attenuators modulated by the RET/PTC1 oncogene. Each row 581

represents one probe set corresponding to one gene. Different colors in the rectangles represent 582

different levels of MAS 5-derived signals after per chip and per gene normalization (Gene Spring, 583

Agilent Technologies, Palo Alto, CA). NT, uninfected thyrocytes; RET/PTC1-Y451F and RET/PTC1, 584

RET/PTC1-Y451F-and RET/PTC1-infected thyrocytes, respectively. (B) real-time RT-PCR analysis of 585

the ERK pathway attenuators genes DUSP4, DUSP6 and SPRY2 (boxed) and PLAU and CSF2 genes, 586

as control, in parental and RET/PTC1- or RET/PTC1-Y451F- infected cells. Results are presented as 587

relative quantification (mRNA expression normalized for PGK1 mRNA levels). 588

589

Figure 2. Gene expression levels of ERK pathway attenuators in PTC. Two different datasets were 590

examined. Data are reported as scatter plots of log values and medians. P values were determined by 591

the Mann-Whitney U-test. P<0.05(*), P<0.01(**), P<0.001(***): statistically significant results in 592

comparison with non-neoplastic thyroid samples. (A) INT dataset. DUSP5 and DUSP6 relative gene 593

expression values in PTC (n=34) and non-neoplastic thyroid (n=9) were measured as log2 ratios. The 594

details of this gene expression analysis have been previously reported (Frattini et al., 2004). (B) 595

GSE27155 dataset (Giordano et al., 2005). The relative gene expression values of the MAPK 596

attenuators DUSP5, 6, 4, 10, SPRY2 and SPRED2 in PTC (n=35) and non-neoplastic thyroid (n=4) 597

were measured as log10 values. 598

599

Figure 3. DUSP6 upregulation via the ERK1/2 pathway by PTC-related oncogenes, DUSP6 600

overexpression and ERK1/2 pathway activation in PTC cell lines. (A) Left panel: real-time RT-601

PCR analysis of the DUSP6 gene in N-thy-ori3-1 cells transiently transfected with the indicated 602

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27

oncogenes. Right panel: Lysates from corresponding N-thy-ori3-1 transfected cells analysed by WB 603

with the indicated antibodies. (B) Left panel: real-time RT-PCR analysis of the DUSP6 gene in K1 or 604

TPC1 PTC cell lines treated with vehicle or UO126. Right panel: Total protein extracts obtained from 605

corresponding K1 or TPC1 treated cells analysed by WB. (C) Analysis of the MAPK signalling 606

pathway in N-thy-ori3-1 and in selected PTC cell lines. The membranes were then stripped and re-607

probed with the respective anti-protein antibodies. (D) Total, cytoplasmic and nuclear extracts were 608

concomitantly analysed with the indicated phospho-specific antibodies. Anti-NBS1 and anti-vinculin 609

blots are shown as nuclear and cytoplasmic controls, respectively. In all of the WB analyses, an anti-610

vinculin blot is shown as a protein loading control. 611

612

Figure 4. DUSP6 RNA expression in PTC surgical samples. Real-time RT-PCR analysis of the 613

DUSP6 gene. Relative expression values and medians are recorded. Comparisons between two groups 614

were performed with the Mann-Whitney U-test. Three or more groups were analysed with the Kruskal-615

Wallis test with Dunn’s multiple comparison post-test, P<0.05 (*), P<0.01 (**), P<0.001 (***). (A) 616

PTC (n=15) and non-neoplastic thyroids (Thyroid, n=5) from the INT thyroid tissue collection are 617

reported. (B) The same specimens of panel (A) showing PTC grouped according to their genetic lesion. 618

BRAF: BRAF-V600E mutation. TK: RET (closed symbols) or TRK (open symbols) tyrosine kinase 619

rearrangement. Unknown: genetic lesions other than those mentioned above. (C) The same specimens 620

as panel (A) showing PTC classified as primary tumour (Primary) or nodal metastasis (Nodal). (D) The 621

same specimens as panel (A) classified according to tumour staging. pT1: tumours less than 1 cm and 622

limited to the thyroid; pT2: tumours larger than 1 cm but not more than 4 cm in greatest dimension and 623

limited to the thyroid gland; pT3: tumours more than 4 cm and limited to the thyroid; pT4: tumours 624

displaying local extra-thyroid spread; nodal: nodal metastasis. 625

626

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Figure 5. IHC analysis of DUSP6, p-ERK and ERK of representative cases reported in Table 2. 627

Serial sections were immunolabeled with the indicated antisera. Representative tumour areas are 628

shown. Original magnification 100x. (A) IHC analysis of non-neoplastic thyroid and PDTC surgical 629

samples. The indicated numbers (#9, #10, and #13) refer to patients reported in Table 2. (B) IHC 630

analysis of distinct areas of the PTC/PDTC case #7 with different histology and with or without the 631

genetic lesion BRAF-V600E. 632

633

Figure 6. Biological effects of DUSP6 silencing on TPC1 cells. (A) Early tubulogenesis assay 634

performed with untransfected (NT), non-targeting siRNA-transfected or siDUSP6-transfected TPC1 635

cells. Representative images (original magnification 10x) and total length quantifications of branched 636

structures are shown. The data are reported as percentages of the control ± SD. Protein extracts from 637

corresponding TPC1 cells were analysed by WB with the indicated antibodies. Anti-vinculin blots are 638

shown as protein loading controls. (B) TPC1 cell growth evaluated by the SRB proliferation assay 639

performed with untransfected (NT), non-targeting siRNA-transfected or siDUSP6-transfected cells at 640

day 5 from transfection. (C) Migration assay and invasion assay performed with non-targeting siRNA-641

transfected or siDUSP6-transfected TPC1 cells. Assays were performed in triplicate, and cells were 642

counted in adjacent fields (n=10). The data are reported as the average cell number per field ±SD. 643

Representative images of SRB-stained cells are shown below (original magnification 40x). Protein 644

extracts from TPC1 transfected cells were analysed by WB with the indicated antibodies (right panel). 645

P<0.05(*), P<0.01(**), P<0.001(***) by Student's t-test are indicated in the figure. 646

647

Figure 7. Biological effects of DUSP6 silencing on NIM-1 cells. (A) NIM-1 cells untransfected (NT), 648

non-targeting siRNA-transfected (non-targeting), siDUSP6-transfected (siDUSP6), untransfected cells 649

exposed to solvent (DMSO) or to UO126 (UO126) were grown for up to 72 h in medium without FBS. 650

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29

Cell proliferation was evaluated with the SRB assay. Representative growth curves from one 651

experiment are shown (left panel). Each point represents the mean of eight independent replicates ± 652

SD. Lysates from corresponding NIM-1 cells were analysed by WB in the same timeframe with the 653

indicated antibodies (right panel). Apoptosis was evaluated with an antibody specific for cleaved 654

PARP. An anti-vinculin blot is shown as a protein loading control. (B) Apoptosis was further evaluated 655

by counting apoptotic nuclei in adjacent fields (n=10), as condensed and fragmented nuclei upon 656

Hoechst 33341 staining under fluorescent microscope (indicated by arrows). Relative quantification is 657

shown in the right panel; data were expressed as the average percentages ± SD. 658

(C) Migration assay and invasion assay performed with non-targeting siRNA-transfected or siDUSP6-659

transfected NIM-1 cells. Assays were performed in triplicate, and cells were counted in adjacent fields 660

(n=10). The data were reported as average cell numbers per field ±SD. Representative images of SRB-661

stained cells are shown below (original magnification 40x). P<0.05(*), P<0.01(**), P<0.001(***) by 662

Student's t-test are indicated in the figure. 663

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Table 1 Antibodies working dilution and staging procedure for immunohistochemical analysis

Antibody Dilution Staining procedure Positive control

DUSP6 (Abcam) 1:150 Incubation ON 4°C. Development with Streptavidin/HRP TPC1 and B-CPAP cell lines

Anti-p42/44 MAPK (Cell Signaling Technology) 1:25 Incubation ON 4°C. Development with Streptavidin/HRP Breast carcinoma

Anti-Phospho-p42/44 MAPK (Cell Signaling Technology) 1:25 Incubation ON 4°C. Development with Streptavidin/HRP Colon carcinoma

TTF1 Dako (Glostrup, Denmark) 1:100 Incubation 1 hour at 25°C. Development with Ultra Vision LP Large Volume Detection System HRP Polymer Thyroid

All samples were pre-treated for antigen retrieval: 15’ at 95°C with citrate buffer pH6

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Table 2. DUSP6 protein expression in thyroid carcinoma specimens ND= not determined, §= The original specimen from these patients contained areas showing PTC component, pTNM= pathological tumor-node-metastasis staging, Nx= regional lymph nodes cannot be assessed, Mx= distant metastasis cannot be assessed, TTF1= thyroid transcription factor 1, NED= Not evidence of disease.

Case no Age Gender BRAF

genotype Specimen T N M DUSP6 TTF1 Clinical outcome (length of follow-up)

1 - Non neoplastic thyroid - - - Very Low + -

2 - Non neoplastic thyroid - - - Very Low + -

3 47 F BRAF-V600E PTC (Tall Cell v.) pT2 Nx Mx High + NED (5 years)

4 47 F wt PTC (NOS) pT3 N1a Mx High + Lost to follow up

5 31 F wt PTC (Follicular v). pT3 Nx Mx Moderate + NED (5 years)

6 67 F wt PDTC § pT4b N1b Mx Moderate + Progression

(1 year)

7 74 F BRAF-V600E

and wt PDTC § pT4a N1a skeleton High (BRAF-V600E) Moderate (BRAF-wt) + Progression

(1 year)

8 74 F wt PDTC § pT3 Nx Mx High + NED (5 years)

9 81 M wt PDTC pT4b Nx Mx Very Low + Lost to follow up

10 74 M wt PDTC pT4b Nx skeleton Moderate-High + Progression

(1 year)

11 73 M wt PDTC pT3 Nx Mx High + NED (1 year)

12 73 F wt PDTC pT3 Nx

lung skeleton

brain High + NED

(4 years)

13 74 M wt PDTC pT3 Nx skeleton High + Progression

(1 year)

14 54 F ND ATC pT4b N1a Mx Very Low - Died of disease

15 68 F ND ATC § pT4a Nx Mx Very Low - Died of disease

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SPRY2

SPRED2

DUSP4

A

Fig. 1

DUSP4

DUSP5

DUSP6

DUSP10

down upB

uant

ifica

tion

200

250

300

350NT RET/PTC1-Y451F RET/PTC1

DUSP4 DUSP6 SPRY2

Relat

ive Q

u

0

50

100

150

PLAU CSF2DUSP4 DUSP6 SPRY2

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1 3

DUSP5 DUSP6Ass

ion

essi

on

*** ***

Fig. 2

Thyroid PTC-1

0

Thyroid PTC-1

0

1

2

B

Rel

ativ

e ex

pre

Rel

ativ

e ex

pre

Thyroid PTC3.0

3.5

4.0

4.5

Thyroid PTC2.5

3.0

3.5

4.0

4.5

B DUSP5 DUSP6

log1

0 e

xpre

ssio

n

log1

0 e

xpre

ssio

n** **

1.52.02.53.03.54.0

DUSP4

2.0

2.5

3.0

3.5

4.0

DUSP10

log1

0 e

xpre

ssio

n

og10

exp

ress

ion

**

Thyroid PTC1.5

Thyroid PTC2.0

2.5

3.0

3.5

4.0SPRY2

2.0

2.5

3.0

3.5SPRED2

log1

0 e

xpre

ssio

n

log1

0 e

xpre

ssio

n

l

*

Thyroid PTC2.5

Thyroid PTC2.0

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A

αVinculin

αBRAF

αRET

αTRK

αVinculin

αBRAF

αRET

αTRK

2468

10

2468

10

Fig. 3

DUSP

6ela

tive

Quan

tifica

tion

K1 TPC1

DM

SO

UO

126

αpERK1/2

αERK1/2

DM

SO

UO

126

αDUSP6

K1 TPC1

DM

SO

UO

126

αpERK1/2

αERK1/2

DM

SO

UO

126

αDUSP6

αVinculinαVinculin0

Mock RET/TPC1 BRAF-V600E

TRK-T30

Mock RET/TPC1 BRAF-V600E

TRK-T3

0,5

1

1,5

B

0,5

1

1,5

ReDU

SP6

elativ

e Qu

antifi

catio

n

αERK1/2αVinculin

αERK1/2αVinculin

C

DM

SO

UO

126

TPC1

DM

SO

UO

126

K1

00

D

Re

αpERK1/2

αERK1/2

αVinculin

αDUSP6

αpRSK1/2

αRSK1

αpMEK1/2

αMEK1/2

αpERK1/2

αERK1/2

αVinculin

αDUSP6

αpRSK1/2

αRSK1

αpMEK1/2

αMEK1/2

αpERK1/2

αERK1/2

αVinculin

αDUSP6

αNBS

αpERK1/2

αERK1/2

αVinculin

αDUSP6

αNBS

Total Extracts

αVinculinTotal

Extracts

αVinculinNuclearExtracts

CytoplasmicExtracts

NuclearExtracts

CytoplasmicExtracts

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**

A

10

15

C

10

15

antifi

catio

n

10

15

10

15 ***

antifi

catio

n

Fig. 4

Thyroid PTC0

5

10

B 15

10

Thyroid Primary Nodal0

D15

Thyroid PTC0

5

DUSP

6 Rela

tive

Qu

15

1010

Thyroid Primary Nodal0

15

*

* ***

DUSP

6 Rela

tive

Qua

5

5

10

15

5

10

15

5

10

15

5

10

15

**

**

***

*

USP6

Rela

tive

Quan

tifica

tion

USP6

Rela

tive

Quan

tifica

tion

Thyroid BRAF TK UNKNOWN0

Thyroid pT1-2 pT3-4Nodal

0

PrimaryThyroid BRAF TK UNKNOWN

0Thyroid pT1-2 pT3-4

0

DU DU

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Non neoplastic thyroid#2

DUSP6

PDTC#13

PDTC#10

PDTC#9

Fig. 5

A

pERK

1/2

ERK1

/2TTF1

BRAF‐V600EBRAF wtBPDTC #7

DUSP6

RK1/2

ERK1

/2pE

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ANT

Fig. 6

60

80

100

120

140

hed

struc

tures

(% of

NT)

*

2,5

Early branching assay Proliferation SRB assayB

***

Non Targeting

iDUSP6

0

20

40

NT Non Targeting siDUSP6

Bran

ch (

0

0,5

1

1,5

2

NT N T ti iDUSP6

αDUSP6

αpERK1/2

αERK1/2

Proli

ferati

on(O

D 55

0nm)

siDUSP6 NT Non Targeting siDUSP6

αVinculin

αpAKT

αAKT

NT Non

eting

SP6

αcleaved PARP

αPARP

800

CMigration assay

d

Invasion assay

eld200 NT Non T

arge

ting

siDUS

P6

NTa

rge

siDUS

0

200

400

600

TPC1

migr

ating

cells

/field

TPC1

inva

dingc

ells/f

ie

0

100

200

** ***

N N

αDUSP6

αpERK1/2

αERK1/2

αpMEK1/2

αMEK1/2

0Non targeting siDUSP6


αVinculin

αpSHC

αSHC

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A

48 h 72 h

NT

Non

Tar

getin

g

siD

US

P6

NT

Non

Tar

getin

g

siD

US

P6

αDUSP61.82

NT

Fig. 7

αDUSP6

DM

SO

UO

126

Proliferation SRB assay

αcleavedPARP

αPARP

αDUSP6

αAKT

αpAKT

αpERK1/2

ERK1/20 20.40.60.8

11.21.41.6

Prol

ifera

tion

(OD

550

) DMSO

UO126

Non Targeting

siDUSP6

***αpERK1/2

α ERK1/2

αAKT

αpAKT

αcleaved PARP

ells/f

ield ***

10

15

αVinculin

αERK1/20

0.2

0h 24h 48h 72h

α ERK1/2

αVinculin

B

Non Targeting

siDUSP6

% of

apop

totic

ce

0

5

10

Non Targeting siDUSP6

C

100

200

300C

1 in

vadi

ngce

lls/fi

eld Invasion assay

200

400

600

800 Migration assay

1mig

ratin

g ce

lls/fi

eld

***


NIM

-


NIM

-1

***

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dusp6/mkp3 is over-expressed in papillary and poorly differentiated ...

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