Expression and Clinical Significance of SHP2 in Gastric Cancer

Abstract

OBJECTIVES:

To investigate the expression and clinical significance of the protein tyrosine phosphatase, nonreceptor type 11 (PTPN11 or SHP2) gene, which encodes Src homology 2 domain-containing phosphatase (SHP-2) in gastric cancer.

Methods:

SHP2 expression was detected by immunohistochemical staining and realtime quantitative reverse transcription- polymerase chain reaction in tissue samples of normal gastric mucosa and different grades of gastric cancer. Correlation between SHP2 expression and standard clinico pathological parameters was analysed.

Results:

Immunohisto - chemical staining revealed significantly higher rates of SHP2 expression in gastric cancer tissues (72.5%) versus normal gastric mucosa (21.9%). SHP-2 mRNA levels were also significantly higher in gastric cancer tissues versus normal gastric mucosa. SHP2 expression correlated significantly with tumour differentiation, clinical classification and lymph node metastases, but was independent of sex and age.

Conclusions:

SHP-2 is upregulated in gastric cancer and may be related to the development of gastric cancer. SHP-2 may be a potential prognostic marker of, or a therapeutic target for, gastric cancer.

Introduction

Src homology 2 domain-containing phosphatase (SHP-2, encoded by the protein tyrosine phosphatase, nonreceptor type 11 [PTPN11], or SHP2, gene) is a widely found cytoplasmic tyrosine phosphatase with two Src homology 2 domains. ¹ Tyrosine phosphorylation is controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), which regulate numerous cellular processes. Some PTPs function as tumour suppressors whereas others are oncogenic, ² and SHP-2 is a PTP that contributes to oncogenesis. ³ Studies have clearly demonstrated that SHP-2 plays important roles in signal transduction from the cell surface to the nucleus and in regulating cytokines and growth factors, thereby influencing cell survival, proliferation and differentiation.^{4 – 7} Mutations in the SHP2 gene may lead to leukaemia.^8,9 Moreover, SHP-2 may be involved in tumorigenesis in liver, bladder or breast cancer.^{10 – 13}

Src homology 2 domain-containing phosphatase functions downstream of cytoxin-associated antigen A (CagA).^{14 – 16} CagA is produced by some strains of Helicobacter pylori and may cause gastric carcinoma: ¹⁷ infection with H. pylori in the presence of CagA has been shown to increase the risk of developing gastric cancer. ¹⁸ It is currently unknown whether SHP-2 is involved in gastric cancer and whether the expression of SHP2 could be a suitable prognostic marker.

While mortality from gastric cancer is in slight decline worldwide, it shows an increasing trend in China, where it is a leading cause of death.^19,20 As early discovery of tumorigenesis or tumour metastases in the stomach can extend the survival of patients, an effective prognostic marker for gastric cancer is urgently needed.

The present study evaluated SHP2 expression as a potential prognostic marker for gastric cancer, the hypothesis being that SHP-2 is related to gastric cancer at some level. Expression of SHP2 in samples of gastric cancer tissue and normal gastric mucosa was assessed using immuno -histochemistry and real-time quantitative reverse transcription–polymerase chain reaction (qRT –PCR).

Patients and methods

Study Population and Tissue Samples

Surgically resected specimens were collected from consecutive patients with gastric cancer who had undergone potentially curative tumour resections at The Department of Gastroenterological Surgery, The First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou, China, between January 2002 and January 2006. These specimens were collected from patients who had not received radiation treatment, chemotherapy or biological therapy for cancer before resection. During the corresponding period, normal gastric mucosa tissue specimens were collected from age-matched patients at autopsy: these patients had died from other causes (e.g. bacterial infection, cardiovascular disease or accident). All autopsies were performed within 3 h of death, and the specimens of normal gastric mucosal tissue underwent all of the same tests as the gastric cancer tissue specimens.

The presence of tumours was confirmed in specimens of gastric cancer tissue by histopathological examination using haematoxylin and eosin staining. All tumours were graded according to the degree of architectural differentiation and cellular features. Histological typing was performed using the World Health Organization 1990 criteria, ²¹ grading tissues as having high, moderate or poor levels of differentiation. Tumour–node–metastasis (TNM) stages were classified according to Union for International Cancer Control (UICC) 2010 TNM classification. ²² Patients with gastric cancer were subsequently observed for ≤ 48 months, to ascertain survival rate.

All procedures in this study were approved by the Ethics Review Committee of The First Affiliated Hospital of Zhejiang University. Tissue samples were collected from patients after having obtained written consent prior to undergoing surgery.

Immunohistochemistry for SHP-2 Protein

Immunohistochemistry was performed using a streptavidin–peroxidase immunohisto -chemistry kit (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd, Beijing, China) according to the manufacturer's instructions. Briefly, each specimen of gastric tissue (obtained at resection or autopsy) was fixed in 10% formaldehyde for at least overnight. The specimen was then dehydrated, embedded in paraffin wax and cut into 8-μm thick sections. Sections were deparaffinized and then incubated in 3% hydrogen peroxide solution for 30 min to quench endogenous peroxidise activity. After washing twice with 0.01 mM phosphate buffered saline (PBS; pH 7.4), slides were incubated with 0.01 M citrate solution (pH 6.0) at 95 °C for 10 min, to retrieve epitopes. After cooling to room temperature, slides were rinsed twice with 0.01 mM PBS, then incubated with 0.01 mM PBS containing 10% normal goat serum for 30 min at room temperature and washed again with 0.01 mM PBS. The slides were incubated with a primary antibody, rabbit antihuman SHP-2 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 1 h, then washed twice with 0.01 mM PBS. Next, the slides were incubated with a secondary antibody, biotin-conjugated goat antirabbit immunoglobulin G (1:100 dilution; Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd), at 37 °C for 30 min and washed twice with 0.01 mM PBS, followed by 3,3′-diaminobenzidine (DAKO, Glostrup, Denmark) staining.

Positive slides were scored (at × 400 magnification) according to the method described by Lu et al: ²³ slides were defined as +, ++ or +++ if 10 – 24%, 25 – 50%, or > 50% of the cells stained positive, respectively. Staining of < 10% was considered negative. Each slide was examined by two pathologists; in cases of conflict, the slide was defined by a consensus.

Real-Time QRT–PCR for SHP-2 MRNA

Real-time qRT–PCR was performed to quantify SHP-2 mRNA levels in gastric tissue specimens. The total RNA was isolated from 100 μg of tissue using a homogenizer and TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The quality of RNA in the samples was determined by electrophoresis through agarose gels and staining with ethidium bromide; 18S and 28S RNA bands were visualized using ultraviolet illumination.

Synthesis of cDNA was performed as described previously. ²⁴ Real-time qPCR was performed on an ABI Prism^® 7500 fast thermal cycler (Applied Biosystems, Foster City, CA, USA). Each sample was run in triplicate in a final volume of 25 μl containing 0.3 μl of cDNA, 10 pmol of each primer, and 12 μl of Power SYBR^® Green PCR Master Mix (Applied Biosystems). PCR thermal cycling conditions involved an initial activation step at 95 °C for 30 s followed by 45 cycles of denaturation at 95°C for 5 s and a combined annealing and extension step at 60 °C for 20 s, with a final step for extension at 72 °C for 7 min. The primers used, based on the cDNA sequence of SHP-2 (Entrez gene number NM_002834) and glyceraldehyde-3-phosphate dehydroge -nase (GAPDH, Entrez gene number NM_002046.4) as an internal control, were as follows: SHP-2 mRNA forward primer 5′-TATCCTCTGAACTGTGCAGATCC-3′, reverse primer 5′-TCTGGCTCTCTCGTACAAGAAAA-3′; GAPDH forward primer 5′-TAAGTATG ACTCCACCCACG-3′, reverse primer 5′-CTAGCACCTTCCCAACTA-3′. The cycle number at which logarithmic PCR plots crossed a calculated threshold line (Ct values) was determined and to calculate Δ^Ct values (Δ^Ct = Ct of the target gene minus Ct of the internal control gene). High Δ^Ct values represent low levels of expression.

Statistical Analyses

Statistical analyses were performed using the SPSS^® software package, version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. Data were presented as mean ± SD or n (%) of participants. Between-group differences in real-time quantitative RT–PCR results were evaluated using a Student's t-test. Between-group differences in categorical data were evaluated using the χ²-test. A P value of < 0.05 was considered to be statistically significant.

Results

In total, 40 gastric cancer tissue specimens and 32 normal gastric mucosa tissue specimens were collected and analysed.

Immunohistochemical analysis revealed that SHP2 expression was significantly associated with gastric cancer (P < 0.001) (Table 1). Real-time qRT–PCR analysis demonstrated that the mean ± SD levels of SHP2 mRNA in gastric cancer (3.57 ± 1.33) were significantly higher than in normal gastric mucosa tissue (8.46 ± 3.21) (P < 0.001).

Table 1:

Immunohistochemical staining for Src homology 2 domain-containing phosphatase (SHP-2) in gastric cancer and normal gastric mucosa tissue samples

SHP2 expression	Tissue type		χ2	Statistical significance a
	Gastric cancer n = 40	Normal gastric mucosa n = 32
−	11	25
+	6	5
++	9	2
+++	14	0
Positive rate	29 (72.5)	7 (21.9)	18.225	P < 0.001

Data presented as n samples or n (%) incidence.

a

χ²-test for between-group analyses.

−, cells negative (< 10% staining positive); +, 10 – 24% cells positive for SHP-2, ++, 25 – 50% cells positive for SHP-2; +++, > 50% cells positive for SHP-2. ²³

Statistical analyses showed that SHP2 expression was significantly associated with tumour differentiation, clinical classification of the gastric cancer and presence or absence of lymph node metastases (Table 2). The positive rate in highly differentiated tissue was significantly lower compared with moderately or poorly differentiated tissue (P = 0.017; across-group comparison). Tissue samples were divided into two groups according to their TNM classification: ²² the T1 + T2 group and the T3 + T4 group. The SHP-2 positive rate was significantly lower in the T1 + T2 group versus the T3 + T4 group (P = 0.035). A significantly higher number of gastric cancer patients with lymph node metastases were SHP-2-positive compared with those without lymph node metastases (P = 0.008). SHP2 expression was independent of patient sex and age (Table 2).

Table 2:

Relationship between Src homology 2 domain-containing phosphatase (SHP2) gene expression and clinical/clinicopathological characteristics of gastric cancer

Characteristic	n	SHP-2 immunostaining results				SHP-2 positive samples	χ2	Statistical significance a
		-	+	++	+++
Gender
Male	24	6	5	6	7	18 (75.0)	0.188	NS
Female	16	5	3	3	5	11 (68.8)
Age, years
≥ 60	20	5	5	4	6	15 (68.2)	0.457	NS
< 60	20	6	3	5	6	14 (77.8)
Differentiation levels
High	8	5	2	1	0	3 (37.5)	8.192	P =0.017
Moderate	12	4	4	3	1	8 (66.7)
Poor	20	2	2	5	11	18 (90.0)
Clinical classification 22
T1 + T2	15	7	5	2	1	8 (53.3)	4.422	P = 0.035
T3 + T4	25	4	3	7	11	21 (84.0)
Lymph node metastases
Presence	30	5	6	8	11	25 (83.3)	7.064	P = 0.008
Absence	10	6	2	1	1	4 (40.0)

Data presented as n samples, or n (%) incidence.

a

χ²-test for between-group analyses.

−, cells negative (< 10% staining positive); +,10 - 24% cells positive for SHP-2, ++, 25 - 50% cells positive for SHP-2; +++, > 50% cells positive for SHP-2. ²³

NS, no statistically significant between-group differences P ≥ 0.05.

All 40 patients were followed up for a median of 29 months (range 6 – 48 months); 24 died during the follow-up period. In the SHP-2-positive group (n = 29), patients survived for a median of 16 months with a 3-year survival rate of 17.9% (five of 28). In the SHP-2-negative group (n = 11), the median survival time was 35 months, with a 3-year survival rate of 77.8% (seven of nine patients). One patient in the SHP-2-positive group and two in the SHP-2-negative group were lost to follow-up. Comparative analysis of the 3-year survival rate showed that SHP2 expression in gastric cancer indicated poor prognosis (χ² = 11.159, P = 0.001).

Discussion

Although SHP-2 is known to be involved in oncogenesis, its pathogenic role is not fully understood. ²⁵ The present study showed that SHP-2 was upregulated at the mRNA and protein levels in gastric cancer tissues, which implied its involvement in gastric cancer. Additionally, increased SHP2 expression in gastric cancer was associated with a poor prognosis; the 3-year survival rate was particularly low in the SHP-2-positive group and patients in this group were prone to die from tumour recurrence. Thus, it is possible that SHP2 expression could be used as a prognostic marker for tumour recurrence. The upregulation of SHP-2 in breast cancer cells has been demonstrated previously. ²⁶ As SHP-2 is a multifunctional protein, ¹ the detailed mechanism of its action in gastric cancer is worthy of further research.

After analyses of 40 tumour specimens from patients with gastric cancer, SHP2 expression was shown to be associated with tumour differentiation, clinical classification and lymph node metastases. This suggests that SHP2 expression is strongly related to the aggressiveness of the tumour. Given the positive role of SHP-2 in cell growth, transformation and stem cell survival, ¹ the present study indicated that SHP2 expression could be used as a prognostic marker for malignant tumour and lymph node metastases in gastric cancer. SHP2 expression was not affected by the sex or age of the patient.

Continued tumorigenesis and metastases are long-standing problems for tumour therapy. Early accurate prognosis can help physicians to adopt the most effective therapy. The present study indicated that expression of the SHP2 gene has potential prognostic significance for gastric cancer.

In general, immunohistochemistry is a convenient tool for large-scale tests and gastric tissues are relatively easily obtained from patients for analysis. Thus, detection of SHP2 expression by immunohistochemistry could be a useful tool for determining tumorigenesis or tumour aggressiveness in gastric cancer. The mechanism of action of SHP-2 in tumorigenesis and tumour metastases is worthy of further research, to understand its potential prognostic and therapeutic usefulness of SHP-2 in the future treatment of gastric cancer.