Dig Dis Sci (2010) 55:15651569

DOI 10.1007/s10620-009-0924-z

ORIGINAL ARTICLE

Overexpression of Protein Phosphatase Non-receptor

Type 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 dephosphorylation 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 alteration 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 immunoreactivity. The PTPN11 expression in the cancers was associated with the tubular morphology (versus signet ring cell

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: kimss@catholic.ac.kr
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

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

the advanced gastric cancer type (versus early gastric
cancer 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 signaling protein in the cells might play a role in the cancer
development. Also, the higher expression of PTPN11 in
tubular and intestinal types, where Helicobacter 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 phosphotyrosine 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].

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Dig Dis Sci (2010) 55:15651569

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]. Furthermore, activating PTPN11 mutations have been found in
leukemias, such as juvenile myelomonocytic leukemia,
B-cell precursor acute lymphocytic leukemia, and acute
myelocytic 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 of Helicobacter pylori
in gastric epithelial cells underwent tyrosine phosphorylation by SRC family kinases. Phosphorylated CagA specifically binds and activates PTPN11. Subsequently,
activated PTPN11 by CagA induces sustained intracellular

Table 1 Summary of PTPN11

expression in the gastric
carcinomas according to
clinicopathologic parameters

signal-regulated kinase (ERK) activation that might promote cellular proliferation [1012]. Despite the importance
of PTPN11 activation in gastric tumorigenesis, to date the
data on PTPN11 expression in gastric carcinomas is lacking. 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

Total no. of cases

Intensity of immunostaining
with positive
(n)
immunostaining (%)
0
?1
?2
?3

Mean score P value

63 (100)

44

16

1.29

7 (100)

1.0

Moderately (n = 30)

30 (100)

26

1.1

Poorly (n = 26)

26 (100)

Tissue differentiation
Tubular (n = 63)
Well (n = 7)

11

12

1.69

Signet ring cell (n = 29) 15 (52)

14

15

0.48

total (n = 92)

78 (87)

14

59

16

Intestinal (n = 33)

33 (100)

26

Diffuse (n = 46)

34 (74)

12

25

0.93

Mixed (n = 13)

11 (85)

1.23

EGC (n = 36)

27 (75)

22

0.86

AGC (n = 56)

51 (91)

37

11

1.20

0.00

Laurens classification
1.18

0.036a

EGC versus AGC

TMN staging
I (n = 42)

33 (79)

26

0.93

II (n = 12)

10 (83)

1.04

III (n = 28)

26 (93)

18

1.32

IV (n = 10)

9 (90)

0.95

T1 (n = 35)

27 (77)

22

0.89

T2 (n = 11)

9 (81)

1.14

T3 (n = 37)

34 (91)

25

1.24

T4 (n = 9)

8 (88)

0.94

Positive (n = 46)

40 (86)

27

10

1.20

Negative (n = 46)

38 (83)

32

0.93

0.026

0.201

Depth of invasion
0.133

Lymph node metastasis

The value is a statistical

difference between intestinal
and diffuse type gastric
carcinoma

123

0.61

Distant metastasis
Positive (n = 8)
Negative (n = 84)

7 (87)

1.00

71 (85)

13

53

15

1.07

0.71

Dig Dis Sci (2010) 55:15651569

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 information 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 Table 1.
Immunohistochemistry
Using sections from the TMA sections, immunohistochemistry for PTPN11 was performed using a streptavidinbiotin complex method (LSAB2 kit/HRP) (DAKO, Glostrup, Denmark). Antibodies for PTPN11 (SC-280, dilution
1/100; Santa Cruz Biotechnology, Santa Cruz, CA) was
used as primary antibody. After deparaffinization, heatinduced 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 microwave 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 interpreted 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 replacement of primary antibody with the blocking reagent.

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Statistical Analysis
The relations between the PTPN11 expression and clinicopathologic 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 immunostainings are summarized in Table 1. 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 Table 1). According to the intensity, 3, 16, 59, and 14
cancers showed grade 3, 2, 1, and 0 immunopositivity,
respectively. The immunostaining of PTPN11, when present, 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 immunostaining was positive in 63 (100%) of 63 tubular adenocarcinomas, whereas it was positive only in 15 (52%) of
29 signet ring cell adenocarcinomas (Table 1). 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 adenocarcinomas 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 immunopositivity in the intestinal-type carcinomas was significantly 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 negative), distant metastasis (positive versus negative), and
depth of invasion (T1T4). However, none of the parameters was significantly different among their subgroups
(P [ 0.05) (Table 1).

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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

Discussion

somewhat different data from those from the breast cancers. 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 contrast, PTPN11 expression in the gastric cancers was significantly 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, suggesting a possibility that PTPN11 expression might be
involved in progression of early type of gastric cancer to
advanced one. Together, these data suggest that clinicopathologic meanings of PTPN11 expression could be different 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 adenocarcinomas was higher than that in signet ring cell carcinomas. Similarly, PTPN11 expression in intestinal-type
adenocarcinomas was higher than that in diffuse-type

The aim of the present study was twofold; first, to determine 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, Table 1). 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 acquisition 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 clinocopathologic variables, however, our results showed

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adenocarcinomas. The signet ring cell type adenocarcinoma 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 following chronic inflammation without passing through
intermediate steps of atrophic gastritis or intestinal metaplasia. Furthermore, H. pylori may have a smaller impact
on development of diffuse-type gastric cancers than the
intestinal type [17, 18].
CagA protein from H. pylori is 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 progression 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. pylori
promotes gastric carcinogenesis, especially in intestinaltype 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.

References
1. Ostman A, Bohmer FD. Regulation of receptor tyrosine kinase
signaling by protein tyrosine phosphatases. Trends Cell Biol.
2001;11:258266.
2. Neel BG, Tonks NK. Protein tyrosine phosphatases in signal
transduction. Curr Opin Cell Biol. 1997;9:193204.
3. Li L, Dixon JE. Form, function, and regulation of protein tyrosine
phosphatases and their involvement in human diseases. Semin
Immunol. 2000;12:7584.
4. Mohi MG, Neel BG. The role of Shp2 (PTPN11) in cancer. Curr
Opin Genet Dev. 2007;17:2330.

1569
5. Neel BG, Gu H, Pao L. The Shping news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem
Sci. 2003;28:284293.
6. Blume-Jensen P, Hunter T. Oncogenic kinase signaling. Nature.
2001;411:355365.
7. Tartaglia M, Gelb BD. Noonan syndrome and related disorders:
genetics and pathogenesis. Comprehensive review of Noonan
syndrome and its affiliated disorders, including discussion of
mutational spectrum, phenotypic features and pathogenic mechanisms. Annu Rev Genomics Hum Genet. 2005;6:4568.
8. Tartaglia M, Niemeyer CM, Fragale A, et al. Somatic mutations
in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet.
2003;34:148150.
9. Bentires-Alj M, Paez JG, David FS, et al. Activating mutations of
the noonan syndrome-associated SHP2/PTPN11 gene in human
solid tumors and adult acute myelogenous leukemia. Cancer Res.
2004;64:88168820.
10. Higashi H, Tsutsumi R, Muto S, et al. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science. 2002;295:683686.
11. Yamazaki S, Yamakawa A, Ito Y, et al. The CagA protein of
Helicobacter pylori is translocated into epithelial cells and binds to
SHP-2 in human gastric mucosa. J Infect Dis. 2003;187:334337.
12. Hatakeyama M. Oncogenic mechanisms of the Helicobacter
pylori CagA protein. Nat Rev Cancer. 2004;4:688694.
13. Lee SH, Shin MS, Park WS, et al. Alterations of Fas (Apo-1/
CD95) gene in non-small cell lung cancer. Oncogene.
1999;18:37543760.
14. Zhou X, Coad J, Ducatman B, Agazie YM. SHP2 is up-regulated
in breast cancer cells and in infiltrating ductal carcinoma of the
breast, implying its involvement in breast oncogenesis. Histopathology. 2008;53:389402.
15. Tahara E. Molecular mechanism of stomach carcinogenesis.
J Cancer Res Clin Oncol. 1993;119:265272.
16. Lauren P. The two histological main types of gastric carcinoma.
Diffuse and so-called intestinal type carcinoma. An attempt at
histoclinical classification. Acta Pathol Microbiol Scand. 1965;
64:3149.
17. Martn-de-Argila C, Boixeda D, Redondo C, et al. Relation
between histologic subtypes and location of gastric cancer and
Helicobacter pylori. Scand J Gastroenterol. 1997;32:303307.
18. Trajkov D, Stardelova K, Dimitrova M, Mishevski J, Serafimoski
V. Helicobacter pylori and gastric carcinoma. Prilozi. 2007;
28:3946.
19. Hatakeyama M. Helicobacter pylori CagAa bacterial intruder
conspiring gastric carcinogenesis. Int J Cancer. 2006;119:1217
1223.

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