dusp6/mkp3 is over-expressed in papillary and poorly differentiated
TRANSCRIPT
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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
Page 1 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
2
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
48
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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
186
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
Page 17 of 38
18
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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20
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
578
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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
Page 26 of 38
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
Page 27 of 38
28
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
Page 28 of 38
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
Page 29 of 38
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
Page 30 of 38
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
Page 31 of 38
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
Page 32 of 38
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
Page 33 of 38
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
Page 34 of 38
**
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
Page 35 of 38
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
Page 36 of 38
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
0Non targeting siDUSP6
αVinculin
αpSHC
αSHC
Page 37 of 38
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
***
0Non targeting siDUSP6
NIM
-
0Non targeting siDUSP6
NIM
-1
***
Page 38 of 38