1
2

Expression of Spred2 in the urothelial tumorigenesis of theurinary bladder

3
4

Shinsuke Oda1, Masayoshi Fujisawa1, Li Chunning1, Toshihiro Ito2, Takahiro Yamaguchi1,

5

Teizo Yoshimura1, and Akihiro Matsukawa1*

6
7

1

8

Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8558, Japan.

92Department of Pathology and Experimental Medicine, Graduate School of Medicine,

Department of Immunology, Nara Medical University, Kashihara, 634-8521, Japan.

10
11

Short title: Spred2 in the urothelial tumorigenesis

12
13

*

14

E-mail: amatsu@md.okayama-u.ac.jp

Corresponding author:

1

15

Abstract

16

Aberrant activation of the Ras/Raf/ERK (extracellular-signal-regulated kinase)-MAPK

17(mitogen-activated protein kinase) pathway is involved in the progression of cancer,

18

including urothelial carcinoma; but the negative regulation remains unclear. In the present

19

study, we investigated pathological expression of Spred2 (Sprouty-related EVH1 domain-

20

containing protein 2), a negative regulator of the Ras/Raf/ERK-MAPK pathway, and the21

relation to ERK activation and Ki67 index in various categories of 275 urothelial tumors

22

obtained from clinical patients. In situ hybridization demonstrated that Spred2 mRNA23

was highly expressed in high-grade non-invasive papillary urothelial carcinoma

24(HGPUC), and the expression was decreased in carcinoma in situ (CIS) and infiltrating

25

urothelial carcinoma (IUC). Immunohistochemically, membranous Spred2 expression,

26

important to interact with Ras/Raf, was preferentially found in HGPUC. Interestingly,

27

membranous Spred2 expression was decreased in CIS and IUC relative to HGPUC, while

28

ERK activation and the expression of the cell proliferation marker Ki67 index were29

increased. HGPUC with membranous Spred2 expression correlated significantly with30

lower levels of ERK activation and Ki67 index as compared to those with negative Spred2

31

expression. Thus, our pathological findings suggest that Spred2 counters cancer

32

progression in non-invasive papillary carcinoma possibly through inhibiting the33

Ras/Raf/ERK-MAPK pathway, but this regulatory mechanism is lost in cancers with high34

malignancy. Spred2 appears to be a key regulator in the progression of non-invasive35

bladder carcinoma.

36

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Introduction

38

Bladder cancer is a highly prevalent disease and its incidence is steadily rising worldwide

39[1]. In the United States, bladder cancer is the 4th most incident and 8th most deadly

40

tumor among men [2]. Most of the bladder cancer is urothelial carcinoma arising from41

urothelial epithelium. Evidence indicates that urothelial carcinoma has two distinct

42

clinical subtypes with distinct molecular features at bladder tumor initiation; low-grade

43

tumors (superficial papillary) and high-grade tumors (flat, represented by carcinoma in

44situ) [3, 4]. Low-grade tumors, i.e., papillary urothelial neoplasm of low malignant

45

potential or low-grade papillary urothelial carcinoma, do not easily progress to high-grade

46

papillary urothelial carcinoma or invasive carcinoma [5, 6]. Recently, a comprehensive

47

landscape of molecular alterations in urothelial carcinomas was shown [7]. More than

48

70% of low-grade papillary carcinomas harbor FGFR3 gene mutation [8]. On the other49

hand, flat carcinoma in situ (CIS) often develops to invasive urothelial carcinoma [9, 10],50

in which allelic deletion of the TP53 (p53) and PTEN (tumor-suppressor) [11] and51

retinoblastoma gene (RB, negative cell cycle regulator) [12] is common.

52

In addition to the gain of function gene mutations, extracellular-regulated kinase

53(ERK) plays a crucial role in cancer development and progression [13, 14]. The

54

Ras/Raf/ERK-MAPK (mitogen-activated protein kinase) pathway, one of the55

serine/threonine kinases of MAPKs pathway, is a major determinant to promote cell

56

proliferation, differentiation, and survival, and plays an important role in bladder cancer

57

prognosis [15]. ERK activation was observed in high-grade non-invasive and invasive

58

urothelial carcinoma [16], suggesting that robust ERK activation contributes to urothelial

59

tumorigenesis with a high malignant potential.

60

Signaling pathways are counterbalanced by endogenous inhibitory mechanism(s).

61Spred2 (Sprouty-related, EVH1 domain-containing protein 2) inhibits Ras-dependent

62

ERK signaling by suppressing the phosphorylation and activation of Raf [17]. Ras

63

activation is aberrant in many tumors due to oncogenic mutation of the Ras genes or64

alterations in upstream signaling components [18]. Rational therapies that target the65

Ras/Raf/ERK-MAPK pathway continues to attract much attention for cancer therapy [19].
3

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We have hitherto investigated in different types of murine models and found that Spred2

67

controls inflammation by down-regulating the Ras/Raf/ERK-MAPK pathway [20–29].

68

Interestingly, Spred2 expression is down-regulated in invasive carcinomas such as

69

hepatocellular carcinoma [30, 31] and prostatic adenocarcinoma [32]. Thus, altered

70

Spred2 expression could affect urothelial tumorigenesis by regulating the Ras/Raf/ERK-71

MAPK pathway in bladder cancer. However; the pathophysiological roles of Spred2 in72

bladder cancer tumorigenesis remain largely unknown. In the present study, we examined73

the mRNA and protein expression of Spred2 in a range of human urothelial tumors. Our

74

present findings suggest that endogenous Spred2 affects urothelial cancer progression,75

especially in non-invasive papillary urothelial carcinoma.

76
77

Materials and methods

78

Clinical samples

79

A total of 275 bladder biopsy or resection specimens (transurethral resection and80

cystectomy) during the year 2001-2016 were retrieved from pathology record at

81

Department of Pathology, Okayama University Hospital. The patients who underwent

82

chemotherapy or radiotherapy before the resection were not included in this study. All the83

hematoxylin and eosin (HE)-stained sections were reviewed and categorized by two

84

blinded pathologists according to the 2016 WHO classification: non-tumor urothelium

85(non-tumor), papillary urothelial neoplasm of low malignant potential (PUNLMP), low-

86

grade papillary urothelial carcinoma (LGPUC), high-grade papillary urothelial carcinoma

87(HGPUC), carcinoma in situ (CIS), and infiltrating urothelial carcinoma (IUC). All88

sections were used for immunohistochemistry. For in situ hybridization, sections were89

randomly chosen from each category. Cases for the enrolled 275 patients were shown in

90

Table 1, in which clinicopathological features of each category were noted.

91

The protocol in this study was reviewed and approved by the Ethics Committee,

92

Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical93

Sciences and Okayama University Hospital (1608-009). Informed consent was obtained4

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in the form of opt-out on our website. Those who rejected were excluded. This consent

95

procedure conformed to amended Ethical Guidelines for Clinical Studies provided by

96

Ministry of Health, Labor and Welfare of Japan (May 31, 2015) was approved by the97

Ethics Committee, Okayama University Graduate School of Medicine, Dentistry and98

Pharmaceutical Sciences and Okayama University Hospital.

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100

In situ hybridization

101

A total of 85 samples were randomly selected from 275 samples (Table 1). Paraffin-

102

embedded tissue samples were sectioned at 5-μm-thick, kept on glass slides overnight at

103

45°C and then in situ hybridization was performed using the Affymetrix ViewRNA ISH

104

Tissue Assay kit (QVT0050) and ViewRNA Chromogenic Signal Amplification kit

105(QVT0200) (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s

106

instructions. Human Spred2 probe set was purchased from Thermo Fisher (Affymetrix,

107

Catalog No. VA1-17417-01). Spred2 mRNA expression was stained in red-dot. The total

108

number of red-dot in 100 cells was counted in each sample under microscope by two109

blinded pathologists, and the number of red-dot per cell was calculated.

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111

Immunohistochemistry112

For immunohistochemistry, all 275 specimens were employed (Table 1). Immunostaining

113

for Spred2 was carried out using the Polink-2 plus HRP rabbit with DAB kit (GBI, Bothell,

114

WA, USA), according to the manufacturer’s instructions. In brief, sections (4-µ-thick)

115

were treated by microwave oven in 0.1 M citric acid buffer, treated with 3%H2O2 in

116methanol, blocked with DAKO Protein Block Serum-Free (Dako, Carpinteria, CA, USA),117

and incubated with anti-human Spred2 polyclonal antibody (Proteintech, Rosemont, IL,

118USA). Sections were then incubated with rabbit antibody specific enhancer, followed by

119

the addition of polymer-HRP for rabbit IgG, and visualized using DAB complex. Nuclear

120

counterstaining was performed using hematoxylin. Immunostaining for pERK1/2 (Clone

121

D13.14.4E, Cell Signaling Technology, Danvers, MA, USA) and Ki67 (Clone MIB-1,

122

Dako) was performed on a Ventana Discovery XT automated stainer (Ventana, Tucson,5123

AZ, USA) with using iVIEW DAB Detection Kit (Ventana).

124
125

Evaluation of immunohistochemical staining

126

Spred2 was stained in the cytoplasm (C) or/and membrane (M). Immunoreactivity for

127

Spred2 was classified into 4 groups, according to subcellular localization and staining

128

intensity; C-M-, absent or weak staining intensity in cytoplasm and membrane; C-M+,

129

moderate to strong membranous staining without staining in cytoplasm; C+M-, moderate

130

to strong cytoplasmic staining without membranous staining; C+M+, moderate to strong131

cytoplasmic and membranous staining. pERK immunostaining was scored on the132

following semiquantitative scale as previously reported with modifications [33]: no133

staining (0); focal to <10% of cells (1); 10-50% of cells (2); 50% or more cells stained

134

weak (3); 10-50% stained strong (4); 50% or more stained strong (5). Ki67 index, a135marker for cell proliferation, was determined by counting 500 tumor cells, and the136

percentages of positively stained cells were determined. The stained sections were137

assessed by two blinded pathologists.

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139

Database analysis

140

Datasets with more than 25 samples in each category from Sanchez-Carbayo bladder 2141[34], Blaveri bladder 2 [35], and Stransky bladder [36] were used to analyze Spred2

142

expression in bladder cancer. An unpaired two-tailed t test was used for the statistical143

analysis. Kaplan-Meier Plotter (http://www.kmplot.com) was used to analyze the144

prognostic values of Spred2 mRNA expression levels in bladder carcinoma. Kaplan-

145

Meier survival plots were drawn using data from the Kaplan-Meier database. A log-rank

146

p-value <0.05 was considered to indicate a statistically significant difference.

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Statistical analysis

149

Statistical analysis was performed using GraphPad Prism7 (GraphPad Software, San

150

Diego, CA, USA) and js-STAR (free software). Dunn's multiple comparison test was

151

performed after Kruskal-Wallis test for the comparison of mean values among multi6

152

groups. Multiple Fisher's exact test was performed using the Bonferroni correction for the153

comparison of proportions among multi-groups. Mann-Whitney test was used for the154

comparison of mean values between the two groups. A value of p<0.05 was considered155

statistically significant.

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157

Results

158

Spred2 mRNA expression in bladder urothelial tumors

159

We first examined Spred2 mRNA expression in various categories of 85 urothelial lesions

160

including non-tumor, PUNLMP, LGPUC, HGPUC, CIS, and IUC. Figure 1A shows the161

representative HE and in situ hybridization photographs from each category, in which

162

Spred2 mRNA expression was presented by red-dot (Fig 1A). The number of red-dots per

163

cell was regarded as Spred2 mRNA expression level (Fig 1B). Levels of Spred2 mRNA

164

expression were increased as the malignancy of the cancer increased in papillary tumors.

165

Of note, the level reached the peak in HGPUC and then decreased in CIS and IUC. Spred2

166

mRNA expression in IUC was significantly lower than that in HGPUC (Fig 1B). These

167

results indicate that Spred2 mRNA expression was up-regulated in non-invasive papillary

168

bladder cancer as compared to cancers with high malignancy including invasive

169

carcinoma.

170
171

Spred2 protein expression in bladder urothelial tumors

172

We next examined Spred2 protein expression by immunohistochemistry in 275 bladder

173

urothelial tumors. To confirm immunoreactivity of Spred2 antibody, H1993 cells were174

stained with the antibody under overexpressing Spred2 (S1 Fig). Spred2 protein

175

expression (Fig 2A) was immunophenotypically classified into 4 groups, according to the176

subcellular localization and staining intensity. The staining pattern in each tumor category177

was shown in Table 2. In all non-tumor cases, Spred2 was positive in cytoplasm of basal

178

and lower intermediate cells (pattern C+M-, 101/101 cases). More than half of the cases

179

of PUNLMP, CIS, and IUC showed absent or weak staining (C-M-; 74% (14/19 cases),180

74% (29/39 cases), and 69% (22/32 cases), respectively). LGPUC and HGPUC showed7

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membranous staining (C-M+ and C+M+) more frequently (49% (20/41 cases), and 51%

182(28/43 cases), respectively) than other categories (Table 2). We then compared mRNA183

and protein expression of Spred2. Cases with membranous staining, regardless of184

cytoplasmic staining pattern (C-M+ and C+M+), showed significantly higher levels of

185

Spred2 mRNA expression than those without membranous staining (C-M- and C+M-) (Fig

186

2B). Spred2 is a membrane-associated substrate of receptor tyrosine kinase [17, 37] and187

react with Raf localized in the raft domain of the plasma membrane [38], suggesting that188

membranous Spred2 is more meaningful when considering the functional regulation. The

189

positive rate of membranous Spred2 expression (C-M+ and C+M+) in each category was

190

shown in Figure 2C, which showed that the expression was increased in LGPUC, peaked191

in HGPUC and then decreased in CIS and IUC. Together with the mRNA expression data,

192

these results suggest that functional Spred2 was most highly expressed in HGPUC, and193

the expression was lower in CIS and IUC as compared to HGPUC.

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Expression of pERK and Ki67 in bladder urothelial tumors

196

Increased Spred2 expression may affect the activation of the Ras/Raf/ERK-MAPK

197

pathway and subsequent cancer growth. To address this possibility, we investigated the198

protein expression of phosphorylated ERK (pERK), an indicator of ERK-MAPK

199

activation status, immunohistochemically in each category. pERK was detected in the200

nucleus and cytoplasm of urothelial epithelial lesions in all specimens from each category

201

with different intensity in strength (Fig 3A). The intensity of nuclear and cytoplasmic

202

staining of pERK was then evaluated, which showed that pERK score in each category

203

increased with increasing malignant potential (Fig 3B). Table 3 summarized pERK score

204

in each Spred2 immunostaining pattern. Weak pERK staining (score, 1 and 2) was

205

detected in 87% (score 1; 67/101, score 2; 21/101 cases) and 100% (score 1; 13/19, score206

2; 6/19 cases) of non-tumor and PUNLMP, respectively. In cancer categories (LGPUC,

207

HGPUC, CIS and IUC), the number of cases with moderate (score 3) and strong (score 4

208

and 5) staining increased. Strong pERK staining was detected in 10% (score 4; 4/41, score209

5; 0/41), 44% (score 4; 14/43, score 5; 5/43 cases), 56% (score 4; 14/39, score 5; 8/39
8

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cases), and 78% (score 4; 16/32, score 5; 9/32 cases) in LGPUC, HGPUC, CIS and IUC,

211

respectively (Table 3). Figure 3C demonstrated the relation between Spred2

212

immunostaining pattern and pERK score for all cases in each category. We next

213

performed Ki67 immunostaining (Fig 4A) and calculated Ki67 index (Fig 4B), an

214

indicator of cell proliferation marker, in all categories. Ki67 index was significantly

215

increased in all categories of bladder urothelial tumors as compared to non-tumor. Ki67

216

index of HGPUC, CIS and IUC was significantly higher than that of LGPUC (Fig 4B).

217

Figure 4C demonstrated the relation between pERK and Ki67 index for all cases in this218

study. Thus, pERK score and Ki67 index increase with increasing malignancy.

219

221

Correlation between pERK score/Ki67 index and membranous
Spred2 expression

222

We next compared the correlation between pERK score/Ki67 index and membranous

223

Spred2 expression (negative: M-, positive: M+) in cancer categories. In HGPUC, pERK

224

score with Spred2 M+ were lower than those with Spred2 M-. No differences were found225

in LGPUC, CIS and IUC (Fig 5A). Since an increase in pERK score is generally

226

associated with an increased Ki67 index [39], ERK activation may result in increased

227

tumor cell proliferation. As shown in Figure 5B, Ki67 index in HGPUC with Spred2 M+

228

was lower than those with Spred2 M-. These results suggest that membranous Spred2

229

plays a role in down-regulated ERK activation and subsequent cancer cell proliferation230

in HGPUC, but this negative regulatory mechanism is not functioning in CIS. Although231

pERK score was not different between Spred2 M- and Spred2 M+ in IUC, Ki67 index

232

was decreased in Spred2 M+ as compared to Spred2 M-, suggesting that Spred2 may

233

downregulate cancer cell proliferation through a mechanism independent of ERK-MAPK

234

in IUC.

220

235
236

Database analyses of Spred2 expression and overall survival

237

We examined Spred2 mRNA expression in bladder cancer database by Sanchez-Carbayo

238

bladder 2 dataset [34], Blaveri bladder 2 [35], and Stransky bladder [36] in a public cancer
9

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microarray database (ONCOMINE) [40]. As shown in Figure 6A, Spred2 mRNA

240

expression was significantly increased in non-invasive superficial bladder cancer

241

compared to that in normal bladder samples (Fig 6A, left). Of note, Spred2 mRNA

242

expression in infiltrating bladder urothelial carcinoma was lower than that in superficial

243

bladder cancer in all three datasets (Fig 6A, middle and right). These data were consistent244

with that in Figure 1B, which showed that Spred2 mRNA expression was upregulated in

245

non-invasive papillary bladder cancer as compared to invasive carcinoma. The decreased

246

Spred2 mRNA expression in infiltrating bladder urothelial carcinoma may have affected247

cancer survival. We then assessed the prognostic value of Spred2 in patients with bladder248

carcinoma by Kaplan-Meier Plotter (www.kmplot.com). The overall survival for 30

249months was higher in patients with higher Spred2 mRNA level (Fig 6B). Although there250

was no statistical significance in the 150 month-overall survival between the groups (Fig251

6C, upper panel), the median survival in high Spred2 mRNA expression group (42.33

252

months) was 1.6 times longer than low expression group (28.63 months) (Fig 6C, lower

253

panel). Thus, the expression level of Spred2 mRNA can be clinically important for the254

prognosis of bladder cancer patients.

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256

Discussion

257

Cancer cell growth is mediated by various cell signaling pathways. Among them, the258

Ras/Raf/ERK-MAPK pathway is often activated in human diseases including cancer [41],259

and as such represents an attractive target for the development of anti-cancer drugs [19].

260This pathway is also important in urothelial cell migration and invasion [42]. A better

261

understanding of the endogenous negative regulatory mechanism(s) could improve262

strategies for preventing and treating bladder urothelial tumors. To the best of our263

knowledge, this is the first report to show Spred2 mRNA and protein expression in

264

bladder urothelial tumors in all categories, ranging from non-tumor to invasive cancer.

265

Previous studies demonstrated that Spred2 mRNA expression was decreased in

266

hepatocellular carcinoma (HCC) [31] and prostatic adenocarcinoma [32], comparing with

267

that in adjacent non-tumor tissue and benign gland, respectively. Down-regulated Spred2
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expression was particularly evident in higher grade prostate cancers [32], and Spred2

269

expression levels in HCC tissue were inversely correlated with the incidence of tumor

270

invasion and metastasis [31]. These previous findings suggested that Spred2 function as271

a potential tumor suppressor gene. In our study, Spred2 mRNA expression was increased272

in non-invasive cancer HGPUC, whereas the expression in invasive bladder cancer IUC

273

was significantly decreased as compared to that in non-invasive carcinoma HGPUC.

274

Consistently, database analyses showed that Spred2 expression in infiltrating bladder275

urothelial carcinoma (invasive) was lower than that in superficial bladder cancer (non-276

invasive). Protein expression of functional Spred2, a membranous positive staining, was277

frequently observed in LGPUC and HGPUC, but not in CIS and IUC. Thus, Spred2 may

278

play a role as a tumor suppressor in non-invasive carcinomas, but the function appears to279

be lost in invasive carcinoma.

280

Spred2 was discovered as a membrane-associated substrates of receptor tyrosine

281

kinases [17, 37]. However; our data indicated that Spred2 was found not only in the282

membrane but in the cytoplasm in urothelial tumors. Previous confocal microscopy

283

analyses revealed that Spred2 was present in cytoplasm and co-localized with neighbor284

of BRCA1 (NBR1) [43] or microtubule-associated protein 1A/1B-light chain 3285(LC3) [44]. Very interestingly, Spred2-NBR1 complex enhanced Spred2-mediated ERK

286

inhibition upon stimulation with fibroblast growth factor (FGF), suggesting that

287

Spred2/NBR1-dependent down-regulation of ERK-MAPK is achieved via directed288

endosomal trafficking of activated receptors [43]. Sprouty proteins, members of the289

Sprouty/Spred family, were distributed throughout the cytosol, which underwent rapid

290

translocation to membrane ruffles following epidermal growth factor (EGF) stimulation

291[45]. In urothelial tumors, we showed that membranous Spred2 protein was often detected292

in cancer categories, especially LGPUC and HGPUC. These results suggest that Spred2

293

translocates from cytoplasm to cell membrane by various stimuli in the cancer

294

microenvironment, exerting its function as a negative regulator of ERK-MAPK.

295

ERK activation was associated with increased Ki67 expression in salivary gland296

mucoepidermoid carcinoma [39]. Since Spred2 inhibits the ERK pathway and subsequent
11

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cell proliferation, we compared the relationship between membranous Spred2 protein

298

expression and pERK score/Ki67 index in each cancer category. Interestingly, HGPUC

299

displaying membranous Spred2 expression showed significantly lower pERK score and300

Ki67 index, as compared to other categories with membrane-negative expression. On the301

other hand, pERK score was not affected by the expression pattern of Spred2 expression

302

in CIS and IUC. Spred2 is presumed to be effective only after reaching a certain level of303

membrane expression. It appears that ERK activation was so strong in CIS and IUC that304

concurrent membranous Spred2 expression might be insufficient to suppress the aberrant

305

ERK activation in CIS and IUC. Spred2 gene mutations can be frequently seen in bladder306

urothelial carcinoma (S2 Fig). Spred2 in CIS and IUC can be mutated, which may fail to307

modulate Ras-Raf interaction. Alternatively, mutational activation of the MAP kinase308

pathway including Ras/Raf is occasionally found in many types of cancer including309

urothelial carcinoma [46, 47]. Spred2 suppresses Ras signaling by preventing

310

the phosphorylation and activation of Raf [17]. Mutated Ras/Raf may fail to interact with

311

Spred2. Interestingly, Ki67 index was decreased in IUC with membranous Spred2

312

expression, although pERK score was not altered by membranous Spred2 expression.

313

Spred2 may downregulate cancer cell proliferation through an ERK-MAPK independent314

pathway in IUC.

315

Spred2 mRNA expression in CIS was as high as that in HGPUC, however; 75% of

316

CIS showed negative membranous Spred2 staining and only 15% of CIS showed positive317

membranous Spred2 staining. It remains unclear how Spred2 protein expression is318

regulated in CIS. The poor correlations were generally reported between the level of319

mRNA and protein [48, 49]. There are many complicated and varied post-transcriptional

320

mechanisms; post-transcriptional, translational and protein degradation regulation. CIS

321

appears to be the critical turning-point to control the complex regulation. Further study is322

necessary to understand the specific mechanisms regulating Spred2 mRNA and protein

323

expression.

324

In conclusion, Spred2 mRNA and protein expression was up-regulated as the grade

325

increased in non-invasive papillary urothelial carcinomas. Membranous Spred2
12

326

expression in HGPUC, but not in CIS and IUC, correlated significantly with low levels

327

of ERK activity. In bladder cancer, HGPUC is clinically important because tumor grows

328

more quickly and more likely spread, and tumor progression (invasion) was identified in329

40% of all cases [50]. Our present study suggests that Spred2 functions to suppress the330

growth and progression of cancer in non-invasive bladder cancer through suppressing the331

ERK pathway, and this regulatory mechanism no longer functions in invasive bladder332

cancer.

333
334

Acknowledgments

335

We thank Mr. Hiroyuki Watanabe and Yasuharu Arashima for their excellent technical336

assistance.

337
338

Author contributions

339

Conceptualization:

340

Data curation: Shinsuke Oda, Akihiro Matsukawa.

341

Formal analysis: Shinsuke Oda, Akihiro Matsukawa.

342

Funding acquisition: Akihiro Matsukawa.

343

Investigation: Shinsuke Oda, Masayoshi Fujisawa, Li Chunning, Takahiro Yamaguchi.

344

Methodology: Shinsuke Oda, Masayoshi Fujisawa, Toshihiro Ito, Teizo Yoshimura.

345

Project administration: Akihiro Matsukawa.

346

Supervision: Akihiro Matsukawa.

347

Writing – original draft: Shinsuke Oda, Masayoshi Fujisawa.

348

Writing – review & editing; Teizo Yoshimura, Toshihiro Ito, Akihiro Matsukawa

Shinsuke Oda, Akihiro Matsukawa.

349

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18

496

classification cohort from a single academic center. Hum Pathol. 2012; 43: 115-120.

497

19

498

Supporting information

499

S1 Fig. Overexpression of Spred2 in H1993 cells. H1993 cells were transfected with500

Spred2 expression plasmid (OriGene, Rockville, MD, USA) or control plasmid

501(OriGene) using turbofectin 8.0 (OriGene). (A) Spred2 mRNA expressions after

502

transfection with control plasmid (control) or Spred2 expression plasmid (Spred2) were503

analyzed by RT-qPCR (n=3 each). Data is presented as mean ± SEM. *p<0.0001 (unpaired

504

t test). (B) Transfection was carried out on Lab-Tek II Slide (8 Chamber, Electron

505

Microscopy Sciences, Hatfield, PA, USA). The cells were fixed in 95% ethanol and506

immunostained with anti-Spred2 polyclonal antibody using the polymer method (Polink-

507

2 Plus HRP RABBIT with DAB kit, GBI, Bothell, WA, USA). Spred2 positive cells were508

shown in brown. (C) Cell extracts after transfection with control plasmid (control) or

509

Spred2 expression plasmid (Spred2) were immunoblotted with indicated primary510

antibodies (n=3 each). Upper: Representative photos were shown. Lower: Band densities

511

were digitised and semi-quantitated. Data is presented as mean ± SEM. (D) Ki67 mRNA

512

expressions after transfection with control plasmid (control) or Spred2 expression

513

plasmid (Spred2) were analyzed by RT-qPCR (n=3 each). Data is presented as mean ±514

SEM. #p<0.05 (unpaired t test). (E) After transfection with control plasmid (control) or

515

Spred2 expression plasmid (Spred2), the cell proliferation was evaluated by CCK-8 assay

516(n=3 each). Data is presented as mean ± SE. *p<0.0001 (unpaired t test).

517
518

S2 Fig. The mutation of Spred2 in cancers. Data were from TCGA Pancancer Atlas

519

from cBioPortal for Cancer Genomics (https://www.cbioportal.org/results/plots). (A) The

520

Spred2 mutations in different cancer. Bladder cancer is the 3rd place having mutation of

521

Spred2 among cancers. (B) The distribution of mutations on the domain structure of522

Spred2 in bladder urothelial carcinoma.

523

20

524

Figure legends

525
526

Fig 1. Spred2 mRNA expression in urothelial tumors. (A) Representative photographs527

of HE- (original magnification 400×) and in situ hybridization-sections from each528

category are shown. Spred2 mRNA expression was presented by red dots. (B) The number529

of the red-dots per cell was counted under microscope and Spred2 mRNA expression

530

level was shown per one cell from each category (N: non-tumor; n=10, P: PUNLMP;

531

n=10, L: LGPUC; n=15, H: HGPUC; n=18, C: CIS; n=18, and I: IUC; n=14). Data were532

mean ± SEM. #p<0.05, §p<0.01, ¶p<0.001, *p<0.0001 (Dunn’s multiple comparison test).

533
534

Fig 2. Immunohistochemical analyses of Spred2 protein expression in urothelial

535

tumors. (A) Representative photographs of Spred2 immunohistochemistry (original536

magnification 400×) from each category are shown. (B) Expression levels of Spred2

537

mRNA in each Spred2 staining pattern were shown. C; cytoplasm, M; membrane. (C-M-;

538

n=81, C+M-; n=122, C-M+; n=54, C+M+; n=11). Data were mean ± SEM. #p<0.05,

539

§

540

expression in each category was shown (N: non-tumor; n=101, P: PUNLMP; n=19, L:

541

LGPUC; n=41, H: HGPUC; n=43, C: CIS; n=39, and I: IUC; n=32). Data were mean ±542

SEM. #p<0.05, §p<0.01 (Multiple Fisher's exact test).

p<0.01 (Dunn’s multiple comparison test). (C) The positive rate of membranous Spred2

543
544

Fig 3. pERK score in urothelial tumors. (A) Representative photographs of pERK

545

immunohistochemistry (original magnification 400×) from each category are shown. (B)546

pERK staining intensity was evaluated and scored (0-5), and pERK score in each category547

was shown (N: non-tumor; n=101, P: PUNLMP; n=19, L: LGPUC; n=41, H: HGPUC;

548

n=43, C: CIS; n=39, and I: IUC; n=32). Data were mean ± SEM. §p<0.01, ¶p<0.001,

549*

p<0.0001 (Dunn’s multiple comparison test).

550
551

Fig 4. Ki67 index in urothelial tumors. (A) Representative photographs of Ki67

552

immunohistochemistry (original magnification 400×) from each category are shown. (B)
21

553

Ki67 index in each category was shown (N: non-tumor; n=101, P: PUNLMP; n=19, L:

554

LGPUC; n=41, H: HGPUC; n=43, C: CIS; n=39, and I: IUC; n=32). Data were mean ±555

SEM. #p<0.05, §p<0.01, ¶p<0.001, *p<0.0001 (Dunn’s multiple comparison test).

556
557

Figure 5. Comparison between pERK score/Ki67 index and membranous Spred2

558

expression. pERK score (A) and Ki67 index (B) were compared between membranous

559

Spred2 negative (M-) and positive (M+) in cancer categories (LGPUC; n=41 (M-:21,

560

M+:20), HGPUC; n=43 (M-:15, M+:28), CIS; n=39 (N-:33, M+:6), and IUC; n=32

561

(M-:22, M+:10)). Bar in each graph represents median. #p<0.05, §p<0.01 (Mann-Whitney

562test).

563
564

Fig 6. Spred2 expression in overall survival of patients with bladder cancer. (A)565

Statistical analyses of Spred2 expression in normal, superficial bladder cancer

566(superficial) and infiltrating bladder urothelial carcinoma (infiltrating) from 3 different

567

datasets (Sanchez-Carbayo bladder 2, Blaveri bladder 2, and Stransky bladder) were

568

shown. The numbers in parentheses indicates the number of samples. §p<0.01, *p<0.0001

569(unpaired two-tailed t test). (B, C) Kaplan-Meier analysis of the data in www.kmplot.com

570

was used to determine the survival probability for 30 months (B) and 150 months (C) of

571

patients with high or low Spred2 expression, followed by the log-rank test.

572

22

573

Table 1. Cases for the enrolled 275 patients.
cases (%)

number of examinedfeaturesby IHC/ISH

progression

nuclear grade

invasivenessnon-tumor

101 (36.7)

101/10---

PUNLMP

19 (6.9)

19/10

slow

very low

non-invasive

LGPUC

41 (14.9)

41/15

slow

low

non-invasive

HGPUC

43 (15.6)

43/18

quick

high

non-invasiveCIS

39 (14.2)

39/18

quick

high

non-invasive

IUC

32 (11.6)

32/14

quick

high>>lowinvasive574

non-tumor; non-tumor urothelium, PUNLMP; papillary urothelial neoplasm of low malignant

575

potential, LGPUC; low-grade papillary urothelial carcinoma, HGPUC; high-grade urothelial

576

carcinoma,

577

immunohistochemistry, ISH; in situ hybridization.

CIS;

carcinoma

in

situ,

IUC;

578

23

infiltrating

urothelial

carcinoma.

IHC;

579

Table 2. Subcellular immunolocalization of Spred2 in each tumor category
C-M-

C+M-

C-M+

C+M+

total cases

non-tumor

0

101

0

0

101

PUNLMP

14

4

1

0

19

LGPUC

15

6

20

0

41

HGPUC

8

7

22

6

43

CIS

29

4

4

2

39

IUC

22

0

7

3

32

total cases

88

122

54

11

275

580

non-tumor; non-tumor urothelium, PUNLMP; papillary urothelial neoplasm of low malignant

581

potential, LGPUC; low-grade papillary urothelial carcinoma, HGPUC; high-grade urothelial

582

carcinoma,583

immunohistochemistry, ISH; in situ hybridization. C; cytoplasm, M; membrane.

CIS;

carcinoma

in

situ,

IUC;

584

24

infiltrating

urothelial

carcinoma.

IHC;

585

Table 3. pERK score in Spred2 immunostaining pattern
Spred2 pattern pERK score

1

2

3

4

5

total cases

non-tumor

C-M-

0

0

0

0

0

0

C+M-

67

21

13

0

0

101

C-M+

0

0

0

0

0

0

C+M+

0

0

0

0

0

0

C-M-

11

3

0

0

0

14

C+M-

2

2

0

0

0

4

C-M+

0

1

0

0

0

1

C+M+

0

0

0

0

0

0

C-M-

3

4

8

0

0

15

C+M-

1

0

3

2

0

6

C-M+

0

5

13

2

0

20

C+M+

0

0

0

0

0

0

C-M-

0

1

2

4

1

8

C+M-

0

0

2

3

2

7

C-M+

0

4

14

4

0

22

C+M+

0

0

1

3

2

6

C-M-

0

1

11

11

6

29

C+M-

0

0

2

1

1

4

C-M+

0

0

2

1

1

4

C+M+

0

0

1

1

0

2

C-M-

0

0

4

11

7

22

C+M-

0

0

0

0

0

0

C-M+

0

1

1

3

2

7

C+M+

0

0

1

2

0

3

PUNLMP

LGPUC

HGPUC

CIS

IUC

586

non-tumor; non-tumor urothelium, PUNLMP; papillary urothelial neoplasm of low malignant

587

potential, LGPUC; low-grade papillary urothelial carcinoma, HGPUC; high-grade urothelial

588

carcinoma,589

immunohistochemistry, ISH; in situ hybridization.

CIS;

carcinoma

in

situ,

IUC;

25

infiltrating

urothelial

carcinoma.

IHC;