ARTICLE IN PRESS

G Model
LUNG-3075; No. of Pages 6
ARTICLE IN PRESS
2 S. Zhang et al. / Lung Cancer xxx (2008) xxx–xxx
Table 1
Clinicopathological characteristics of 90 NSCLC tumors and PYK2 expression by
immunohistochemistry
Clinicopathological characteristics Cases
(n = 90)
Higher
expression
(n = 58)
Lower
expression
(n = 32)
p-Value
Tumor size and invasiveness
T1+T2 47 34 13 0.102
T3+T4
Histological type
43 24 19
Sq 48 27 21 0.083
Ad
Differentiation
42 31 11
Well 37 25 12 0.605
Poor–moderate
Stage
53 33 20
I + II 35 17 18 0.012 *
III + IV
Lymph node status
55 41 14
+ 48 37 11 0.007 **
− 42 21 21
PYK2 expression: * significant difference between early (I + II) and advanced (III + IV)
stage of NSCLCs; ** significant difference between tumors with positive (+) and negative
(−) nodes.
migratory ability of tumor cells, which is supported by the high
activity of Tyr402 found in the progression of breast tumor cells to
have invasive and metastatic phenotype [15].
However, despite the increasing emphasis on PYK2 in human
tumors, whether it positively participates in primary human nonsmall
cell lung cancer has not yet been determined. The aim of
this study is to investigate the expression and clinical significance
of PYK2 in 90 surgically resected NSCLCs with different clinicopathological
features, and the association of PYK2 expression and
activation with ERK1/2 activity and metastatic potential of NSCLC
cell lines.
2. Materials and methods
2.1. Tissue samples and patients
A total of 90 cases of non-small cell lung tumors were retrospective
database from the Pathology Department of China
Medical University. All of the enrolled patients underwent curative
surgical resection without having chemotherapy or radiation therapy.
Formalin-fixed paraffin-embedded sections of tumor obtained
from surgical samples were stained routinely with hematoxylin and
eosin (H&E), and reviewed by two senior pathologists in order to
determine the histological type and stage, according to the WHO
classification of lung and pleural tumors (2004) and the TNM staging
system (1997). 30 cases (included in the 90 cases) of tumor
and paired non-tumorous portion (distant from the primary tumor)
of the same case were quickly frozen in deep freeze refrigerator
until protein extraction. Lymph node status was determined by
routine pathological examination of dissected pulmonary hilar and
mediastinal and intrapulmonary lymph nodes. Clinicopathological
information of the patients about tumor size, histological type, differentiation,
stage and lymph node metastasis was obtained from
patient records, and summarized in Table 1.
2.2. Immunohistochemistry
90 paraffin sections of tumor were deparaffinized and rehydrated
routinely. The slides were then heated in an autoclave
sterilizer for 2 min in 0.1 mol/L Tris–HCl buffer at pH 10. The sections
were incubated overnight with primary rabbit polyclonal antibody
detecting PYK2 (1:100 dilution, sc-9019) (Santa Cruz Biotechnology),
following 3% H 2O 2 and 5% rabbit serum treatment at 37 ◦ C
for 1 h. After which they were incubated with second antibody and
SP complex for 30 min (SP kit-9710), lastly they were visualized
with DAB (DAB kit-0031) (both from Maixin Biotechnology). Hepatocellular
carcinoma tissues were used as positive controls for
PYK2, and negative controls were prepared by non-immune rabbit
IgG at the same dilution as for the primary antibody. All the
immunoreactions were separately evaluated by two senior pathologists.
Brown particles appearing in cytoplasm was as regarded
as positive cells. The intensity of PYK2 immunostaining (1 = weak,
2 = moderate, and 3 = intense) and the percentage of positive tumor
cells (0% = negative, 1–50% = 1, 51–75% = 2, ≥76% = 3) were assessed
in at least 5 high power fields (×400 magnification). The scores of
each tumorous sample were multiplied to give a final score of 0, 1,
2, 3, 4, 6 or 9, and the tumors were finally determined as negative:
score 0; lower expression: score ≤4; or higher expression: score
≥6.
2.3. Western blotting
Frozen tissues (including tumor and non-tumorous portion)
or cells were washed twice with ice-cold phosphate-buffered
saline (PBS), homogenized on ice in 10 volumes (w/v) of lysis
buffer containing 20 mM Tris–HCl, 1 mM EDTA, 50 mM NaCl,
50 mM NaF, 1 mM Na 3VO 4, 1% Triton-X100, 1 mM PMSF and phosphatase
inhibitor using a homogenizer (Heidoph, DLA × 900). The
homogenate was centrifuged at 15000 rpm for 30 min at 4 ◦ C. The
supernatant was collected and stored at −70 ◦ C. Protein content
was determined by the BCA assay (BCA protein assay kit-23227,
Pierce Biotechnology). From each sample preparation, 80 �goftotal
protein was separated by 8% SDS-PAGE and then transferred to
PVDF blotting membranes. The total protein extracts were analyzed
by immunoblotting with indicated antibodies following SDS-PAGE
analysis. Immunoblots were performed using rabbit polyclonal
primary antibodies specific for PYK2, p-Tyr402 and �-actin (a
housekeeping protein used as a loading control to assure equal
amounts of protein in all lanes), and mouse monoclonal antibody
for p-ERK1/2. After blocking nonspecific binding with 5%
BSA in TBS (pH 7.5) containing 0.05% Tween-20 (TBST), primary
antibodies were incubated on the membranes for PYK2 (1:300,
sc-9019), p-Tyr402 (1:200, sc-9023), p-ERK1/2 (1:200, sc-7383)
and �-actin (1:200, sc-1616-R) (all from Santa Cruz Biotechnology)
overnight at 4 ◦ C in TBST. Following three times washes
in TBST, the membranes were incubated for 2 h at 37 ◦ C with
secondary goat anti-rabbit IgG antibodies (1:2000, ZDR-5306)
and goat anti-mouse IgG antibody (1:2000, ZDR-5307) labeled
with horseradish peroxidase (all from Zhongshan Biotechnology).
Immunoreactive straps were identified using the DAB system (DAB
kit-0031, Maixin Biotechnology), as directed by the manufacturer.
Specific bands for PYK2, p-Tyr402, p-ERK1/2 and �-actin were identified
by prestained protein molecular weight marker (SM0441,
MBI Fermentas). The EC3 Imaging System (UVP Inc.) was used
to catch up the specific bands, and the optical density of each
band was measured using an Image J software. The ratio between
the optical density of interest proteins and �-actin of the same
sample was calculated as relative content and expressed graphically.
2.4. Cell lines and culture
Lung PG (pulmonary giant) cell lines including the higher
metastatic BE1 cells and the lower metastatic LH7 cells [16] were
Please cite this article in press as: Zhang S, et al., Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer, Lung Cancer (2008),
doi:10.1016/j.lungcan.2008.05.008