overexpression of protein phosphatase non-receptor type 11 (ptpn11) in gastric carcinomas

7/24/2019 Overexpression of Protein Phosphatase Non-receptor Type 11 (PTPN11) in Gastric Carcinomas

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O R I G I N A L A R T I C L E

Overexpression of Protein Phosphatase Non-receptorType 11 (PTPN11) in Gastric Carcinomas

Jin Soo Kim

Ok Ran Shin

Hyung Keun Kim

Young Seok Cho

Chang Hyeok An

Keun Woo Lim

Sung Soo Kim

Received: 30 March 2009/ Accepted: 16 July 2009 / Published online: 19 August 2009

Springer Science+Business Media, LLC 2009

Abstract

Background Tyrosine phosphorylation and dephospho-rylation by protein tyrosine kinases and phosphatases

(PTPs), respectively, play crucial roles in cellular signal

transduction. Protein phosphatase non-receptor type 11

(PTPN11) is a positive signaling PTP that activates RAS

and ERK signaling. Also, the PTPN11 binds with CagA of

Helicobacter pylori in gastric epithelial cells.

Aim The aim of this study was to explore whether alter-

ation of PTPN11 protein expression is a feature of gastric

cancer cells.

Methods We analyzed PTPN11 expression in 92 gastric

cancer tissues by immunohistochemistry using a tissue

microarray method.

Results The gastric cancers expressed PTPN11 in 78

(87%) specimens, while the epithelial cells in normal

gastric mucosa did not display any PTPN11 immunoreac-

tivity. The PTPN11 expression in the cancers was associ-

ated with the tubular morphology (versus signet ring cell

type), the Laurens intestinal type (versus diffuse type), and

the advanced gastric cancer type (versus early gastriccancer type).

Conclusion Our data indicate that gastric cancers display

a higher expression of PTPN11 protein than the normal

cells, suggesting that neo-expression of this positive sig-

naling protein in the cells might play a role in the cancer

development. Also, the higher expression of PTPN11 in

tubular and intestinal types, whereHelicobacter pylori has

a definite role in the development of the cancers, suggest a

possibility that PTPN11 might play a role in regulation in

Helicobacter pylori pathogenesis the gastric cancers.

Keywords PTPN11

Gastric carcinoma

Expression

Immunohistochemistry

Introduction

Phosphorylation of tyrosine residues is a feature of many

cellular signaling pathways, and is coordinately controlled

by protein tyrosine kinases and phosphatases (PTPs). The

PTPs have either positive or negative role in a variety of

signal transduction pathways [1]. Mammalian PTPs can be

divided into either transmembrane receptor PTPases or

intracellular (non-receptor) PTPases [2, 3]. Non-receptor

PTPs encode two tandem SRC homology 2 (SH2) domains

that enable the binding of the PTPs to specific phospho-

tyrosine residues within protein sequences [4]. Protein

phosphatase non-receptor type 11 (PTPN11), also known

as SHP2, is a positive signaling PTP that is located

downstream of growth factor, cytokine, and extracellular

matrix receptors, and plays crucial roles in regulating cell

growth, transformation, differentiation, and migration [5].

Jin Soo Kim and Ok Ran Shin contributed equally in this study.

J. S. Kim H. K. Kim Y. S. Cho S. S. Kim (&)

Department of Internal Medicine, College of Medicine,

Uijongbu St. Marys Hospital, The Catholic University of Korea,65-1 Gumoh-dong, Uijongbu, Kyunggido, Seoul 480-717, Korea

e-mail: [email protected]

O. R. Shin

Department of Pathology, College of Medicine, Uijongbu

St. Marys Hospital, The Catholic University of Korea,

Seoul, Korea

C. H. An K. W. Lim

Department of General Surgery, College of Medicine, Uijongbu

St. Marys Hospital, The Catholic University of Korea,

Seoul, Korea

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Dig Dis Sci (2010) 55:15651569

DOI 10.1007/s10620-009-0924-z


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Altered expressions and/or mutations in PTKs are linked

to many forms of diseases, including developmental

anomalies and cancers [6]. Recent studies have revealed

that germline activating mutations of PTPN11 cause a

developmental disorder Noonan syndrome [7]. Further-

more, activating PTPN11 mutations have been found in

leukemias, such as juvenile myelomonocytic leukemia,

B-cell precursor acute lymphocytic leukemia, and acutemyelocytic leukemia [8], and to a less extent in solid tumors

[9]. However, a mutational study disclosed that PTPN11

was not somatically mutated in gastric carcinomas [9].

Recent studies showed that CagA ofHelicobacter pylori

in gastric epithelial cells underwent tyrosine phosphoryla-

tion by SRC family kinases. Phosphorylated CagA spe-

cifically binds and activates PTPN11. Subsequently,

activated PTPN11 by CagA induces sustained intracellular

signal-regulated kinase (ERK) activation that might pro-

mote cellular proliferation [1012]. Despite the importance

of PTPN11 activation in gastric tumorigenesis, to date the

data on PTPN11 expression in gastric carcinomas is lack-

ing. In the present study, we analyzed protein expression of

PTPN11 in a series of gastric carcinoma tissues by

immunohistochemistry using a tissue microarray (TMA)

approach.

Methods

Tissue Samples

Ninety-two patients with primary gastric adenocarcinomas

who underwent gastrectomy at Uijongbu St. Marys

Table 1 Summary of PTPN11

expression in the gastriccarcinomas according to

clinicopathologic parameters

a The value is a statistical

difference between intestinal

and diffuse type gastric

carcinoma

Total no. of cases

with positiveimmunostaining (%)

Intensity of immunostaining

(n)

Mean score P value

0 ?1 ?2 ?3

Tissue differentiation

Tubular (n = 63) 63 (100) 0 44 16 3 1.29

Well (n = 7) 7 (100) 0 7 0 0 1.0

Moderately (n = 30) 30 (100) 0 26 4 0 1.1 0.00

Poorly (n = 26) 26 (100) 0 11 12 3 1.69

Signet ring cell (n = 29) 15 (52) 14 15 0 0 0.48

total (n = 92) 78 (87) 14 59 16 3

Laurens classification

Intestinal (n = 33) 33 (100) 0 26 7 0 1.18 0.036a

Diffuse (n = 46) 34 (74) 12 25 8 1 0.93

Mixed (n = 13) 11 (85) 2 8 1 2 1.23

EGC versus AGC

EGC (n = 36) 27 (75) 9 22 5 0 0.86 0.026

AGC (n = 56) 51 (91) 5 37 11 3 1.20

TMN staging

I (n = 42) 33 (79) 9 26 7 0 0.93

II (n = 12) 10 (83) 2 7 3 0 1.04 0.201

III (n = 28) 26 (93) 2 18 5 3 1.32

IV (n = 10) 9 (90) 1 8 1 0 0.95

Depth of invasion

T1 (n =

35) 27 (77) 8 22 5 0 0.89T2 (n = 11) 9 (81) 2 5 4 0 1.14 0.133

T3 (n = 37) 34 (91) 3 25 6 3 1.24

T4 (n = 9) 8 (88) 1 7 1 0 0.94

Lymph node metastasis

Positive (n = 46) 40 (86) 6 27 10 3 1.20 0.61

Negative (n = 46) 38 (83) 8 32 6 0 0.93

Distant metastasis

Positive (n = 8) 7 (87) 1 6 1 0 1.00 0.71

Negative (n = 84) 71 (85) 13 53 15 3 1.07

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Hospital from January 2001 to January 2004 were enrolled

in this study. Ages of the patients ranged 4779 years with

an average of 63 years. None of the patients received

chemotherapy before surgical operation. Clinical informa-

tion of the patients was obtained from medical records.

A TMA recipient block was constructed containing

paraffin-embedded primary tissues from 92 archival patient

specimens, previously fixed in 10% formaldehyde,according to established methods [13]. From every archival

paraffin block, one cylinder from cancer tissues of each

patient of 3.0 mm diameter was taken from representative

areas and transferred to paraffin recipient blocks using a

Tissue Arrayer (Beecher Instruments, Gene Micro-Array

Technologies, Silver Spring, MD). We also included

available lymph node metastasis (n = 14) and distant

metastasis (n = 14) of the cancers in the TMA. As a

control, ten normal gastric mucosal specimens were

included from the patients with chronic gastritis. Other

clinicopathologic data are summarized in Table1.

Immunohistochemistry

Using sections from the TMA sections, immunohisto-

chemistry for PTPN11 was performed using a streptavidin-

biotin complex method (LSAB2 kit/HRP) (DAKO, Glost-

rup, Denmark). Antibodies for PTPN11 (SC-280, dilution

1/100; Santa Cruz Biotechnology, Santa Cruz, CA) was

used as primary antibody. After deparaffinization, heat-

induced epitope retrieval was conducted by immersing

slides in Coplin jars filled with 10 mmol/l citrate buffer

(pH 6.0) and boiling the buffer for 30 min in a pressure

cooker (Nordic Ware, Minneapolis, MN) inside a micro-

wave oven at 700 W; the jars were then cooled for 20 min.

After the epitope retrieval, slides were treated with 0.3%

H2O2 in PBS for 15 min at room temperature to abolish

endogenous peroxidase activity. After washing with TNT

buffer (0.1 mol/l TrisHCl, pH 7.4, 0.15 mol/l NaCl and

0.05% Tween 20) for 20 min, the slides were treated with

TNB buffer (0.1 mol/l TrisHCl, pH 7.4, 0.15 mol/l NaCl

and 0.5% blocking reagent). Sections were incubated

overnight at 4C with the primary antibody. Reaction

products were developed with diaminobenzidine and

counterstained with hematoxylin.

By visual inspection under microscope, we graded the

immunoreactivity as 0, 1, 2, and 3. Tumors were inter-

preted as positive by immunohistochemistry when at least

weak (grade 1) to intense (grade 3) staining was seen in

greater than 30% of the neoplastic cells. Tumors were

interpreted as negative (-) by immunohistochemistry

when no (or very weak) staining was seen in the cells or

when immunostaining was seen in less than 30% of the

cell. As negative controls, a slide was treated by replace-

ment of primary antibody with the blocking reagent.

Statistical Analysis

The relations between the PTPN11 expression and clinico-

pathologic variables were analyzed by the Chi-square test,

Fishers exact test, MannWhiney test and Kruskallwallis

test.P values less than 0.05 were considered significant.

Results

With the immunohistochemical approach using a TMA, we

analyzed the expression of PTPN11 protein in normal

gastric and gastric cancer tissues. The data on the immu-

nostainings are summarized in Table1. Normal foveolar

epithelial cells in the gastric mucosa displayed no PTPN11

immunoreactivity in the specimens analyzed (Fig. 1). In the

gastric carcinomas, immunopositivity (defined as C30% of

the neoplastic cells in more than grade 1 intensity) for

PTPN11 was observed in 78 (87%) of the 92 cancers (Fig. 1

and Table1). According to the intensity, 3, 16, 59, and 14cancers showed grade 3, 2, 1, and 0 immunopositivity,

respectively. The immunostaining of PTPN11, when pres-

ent, was cytoplasmic (Fig.1). A negative control using the

blocking solution instead of the primary antibody showed

no signal (data not shown).

With respect to the histological type, PTPN11 immu-

nostaining was positive in 63 (100%) of 63 tubular ade-

nocarcinomas, whereas it was positive only in 15 (52%) of

29 signet ring cell adenocarcinomas (Table1). There was a

significant difference of PTPN11 immunostaining between

tubular and signet ring cell adenocarcinomas (P\ 0.01).

Moreover, the mean intensity score in the tubular adeno-

carcinomas was significant higher than that in the signet

ring cell adenocarcinomas (1.29 and 0.48, respectively,

P\ 0.01). According to the differentiation of the tubular

adenocarciomas, the mean score of the immunostaining

was significantly higher in poorly differentiated than in

well or moderately differentiated carcinomas (P\ 0.01).

In regard to Laurens classification, PTPN11 immu-

nopositivity in the intestinal-type carcinomas was signifi-

cantly higher than that in diffuse-type carcinomas

(P = 0.036). The immunopositivity in advanced gastric

cancers (AGC) was significantly higher than that in early

gastric cancers (EGC) (P = 0.026). According to the TMN

stages, both highest positive rate and highest mean score

were observed in stage III cancers (93% and 1.32,

respectively), but neither was statistically significant

(P[ 0.05). We also analyzed the PTPN11 expression

according to lymph node metastasis (positive versus neg-

ative), distant metastasis (positive versus negative), and

depth of invasion (T1T4). However, none of the param-

eters was significantly different among their subgroups

(P[ 0.05) (Table1).

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Discussion

The aim of the present study was twofold; first, to deter-

mine whether a PTP protein PTPN11 that plays an

important role in cell growth and proliferation is expressed

in tissue sections of gastric carcinomas, and second, to

determine whether there is any association of its expression

with clinicopathologic parameters. PTPN11 is highly

expressed in the gastric cancer tissues, irrespective of the

pathologic characteristics (Fig.1, Table1). By contrast,

the PTPN11 is negatively expressed in normal gastric

epithelial cells in the mucosa that may be a counterpart of

gastric carcinomas. These results suggest that the acquisi-

tion of oncogenic PTPN11 expression might be involved in

the development of gastric carcinomas. It also suggests that

PTPN11 expression might not be involved in the regulation

of normal cell proliferation in gastric mucosa.

There has been a report on PTPN11 expression in

human cancers. Zhou et al. [14] reported that PTPN11 was

up-regulated in infiltrating ductal carcinomas of breast. In

agreement with this, our data showed that PTPN11 is also

overexpressed in gastric carcinomas. Regarding the clino-

copathologic variables, however, our results showed

somewhat different data from those from the breast can-

cers. The PTPN11 expression in the breast cancers was

positively related to both lymph node metastasis and higher

tumor grades [14], indicating that PTPN11 expression may

be associated with progression of breast cancer. By con-

trast, PTPN11 expression in the gastric cancers was sig-

nificantly related to neither of them (Table 1). Instead, our

data revealed that the PTPN11 expression was positively

related to the advanced type of the gastric cancers, sug-

gesting a possibility that PTPN11 expression might be

involved in progression of early type of gastric cancer to

advanced one. Together, these data suggest that clinico-

pathologic meanings of PTPN11 expression could be dif-

ferent depending on cancer types. To see whether PTPN11

overexpression is a common feature of human cancers, and

whether there are diverse clinicopathologic variables

associated with PTPN11 expression, additional studies on

PTPN11 expression in different types of human cancer

tissues may be required.

We observed that PTPN11 expression in tubular ade-

nocarcinomas was higher than that in signet ring cell car-

cinomas. Similarly, PTPN11 expression in intestinal-type

adenocarcinomas was higher than that in diffuse-type

Fig. 1 Visualization of PTPN11 expression in normal and malignant

gastric mucosal cells by immunohistochemistry. Antibodies were

detected by a diaminobenzidine method. Counterstaining of nuclei

was done with hematoxylin. a In a normal gastric mucosa, the

glandular cells are negative for PTPN11 immunostaining. b A gastric

carcinoma of signet ring cell type shows a negative PTPN11

immunostaining in the cytoplasm. c In a well-differentiated tubular

adenocarcinoma, the cancer cells show a weak (grade 1) PTPN11

immunostaining in the cytoplasm. d In a poorly-differentiated tubular

adenocarcinoma, the cancer cells show a strong (grade 3) PTPN11

immunostaining in the cytoplasm. Original magnifications (ad),

9200

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adenocarcinomas. The signet ring cell type adenocarci-

noma is categorized into diffuse-type gastric cancer, which

has differences in epidemiology, etiology, pathogenesis,

and biologic behavior compared to the intestinal type [15,

16]. The diffuse-type cancer develops in the stomach fol-

lowing chronic inflammation without passing through

intermediate steps of atrophic gastritis or intestinal meta-

plasia. Furthermore, H. pylori may have a smaller impacton development of diffuse-type gastric cancers than the

intestinal type [17,18].

CagA protein fromH. pyloriis delivered into the gastric

epithelial cells and, on tyrosine phosphorylation by Src

family kinases, specifically binds and activates PTPN11

oncoprotein. Activated PTPN11 positively regulates Erk

MAP kinase activity to play an important role in the pro-

gression of G1 to S phase, and induces formation of an

elongated cell shape known as the hummingbird phenotype

[19]. As PTPN11 transmits positive signals for cell growth

and motility, deregulation of PTPN11 by CagA is an

important mechanism by which cagA-positive H. pyloripromotes gastric carcinogenesis, especially in intestinal-

type gastric cancers. Analysis of PTPN11 expression in

both normal gastric and gastric cancer tissues depending on

H. pylori infection status is needed in future studies to

elucidate the exact role of PTPN11 in gastric cancer

development.

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overexpression of protein phosphatase non-receptor type 11 (ptpn11) in gastric carcinomas

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