Download Expression of stem cell self-renew gene, hiwi is a poor prognostic

HIWI expression profile in cancer cells and its prognostic
value for colorectal cancer patients
ZENG Yan1, QU Like1, MENG Lin1, LIU Caiyun1, DONG Bin2, XING Xiaofang1,
WU Jian1 and SHOU Chengchao1*
Running Title: HIWI and early stage of colorectal cancer
1
Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education), Department of Biochemistry and Molecular Biology, Peking University
Cancer Hospital & Institute, Beijing 100142, China.
2
Department of Pathology,
Peking University Cancer Hospital & Institute, Beijing 100142, China.
*Correspondence author:
Chengchao Shou, MD, PhD
Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education)
Department of Biochemistry and Molecular Biology
Peking University Cancer Hospital & Institute
52 Fucheng Road, Haidian District, Beijing 100142, China
Tel: 8610-88196766; Fax: 8610-88122437; E-mail: [email protected]
Grant support:
This work was supported by National Natural Science Foundation of China
(30772489) and National 973 Program (2009CB521805).
Key Words: HIWI; colorectal cancer; prognosis
Abstract
Background: HIWI is a member of PIWI gene family and its expression was found in
various tumors, indicating it may play a pivotal role in tumor development. This study
was designated to examine HIWI expression profile in 31 cell lines and its prognostic
value for colon cancer patients.
Patients and Methods: 270 patients who underwent surgical resection of primary
colorectal cancer between January 1999 and December 2002 with a median follow-up
time
of
33
months
were
registered
in
the
study.
Formalin-fixed
and
paraffin-embedded specimens from these patients and 236 matched adjacent
non-cancerous normal colorectal tissues were collected. Anti-HIWI monoclonal
antibodies were generated and used for evaluating HIWI protein expression in tissues
and 31 cell lines. χ2 tests were conducted to determine the association between HIWI
expression and the other variables. Survival curves were estimated using the
Kaplan–Meier method and compared by the log rank test. Multivariate analysis was
performed by using the Cox regression model.
Results: By generating antibodies specific for HIWI, we examined HIWI protein
expression in several cancer cell lines and demonstrated positive expression of HIWI
in 69 out of 270 (25.6%) colorectal cancer tissues. 15 of 236 (6.4%) matched adjacent
non-cancerous tissues were also positive for HIWI. Patients with positive HIWI
expression in adjacent non-cancerous tissue had statistically lower over survival (OS)
and disease free survival (DFS) compared with negative patients (OS: 10.4% versus
55.5% P=0.009; DFS: 10.4% versus 55.1% P= 0.015). For early stage group (stage I
and II), patients with positive HIWI expression had significantly lower OS and DFS
(OS: 57.4% versus 79.5% P=0.014; DFS: 56.7% versus 80.5% P= 0.010). Patients
with lymph node metastasis had statistically lower OS and DFS (OS: 53.0% versus
73.5% P=0.037; DFS: 52.2% versus 74.6% P= 0.025). Multivariate analysis revealed
that HIWI over-expression was a significant prognostic factor for OS (95%CI：
1.132-2.479，P=0.010).
Conclusion: HIWI could be a potential biomarker for the prognosis of colorectal
cancer patients, especially for those at early stages or without lymph node metastasis.
Introduction
The evolutionarily conserved genes of PIWI family play important roles in stem-cell
self-renewal, RNA silencing and translational regulation in various organisms (1). As
a member of the PIWI gene family, HIWI represents the first class of human genes
known to be required for stem cell self-renewal in diverse organisms (2).
By screening a human testis cDNA library, partial HIWI cDNA was cloned in1998 by
Cox et al, showing 47.1% identical amino acid sequence to the Drosophila PIWI over
its full length, with 58.7% identity at the carboxyl terminus (2). In 2001, Sharma et al.
demonstrated that HIWI gene is exclusively present in human CD34+ haematopoietic
progenitor cells but not in more differentiated cell populations (3). Thereafter,
enhanced HIWI expression was detected in testicular seminomas originating from
embryonic germ cells with retention of germ cell phenotype, but not in
nonseminomas- testicular tumors (4). The expression of HIWI at the RNA level in
primary soft-tissue sarcomas (STS) was also investigated. Taubert et al. reported that
an elevated or reduced HIWI mRNA expression significantly indicated poor
prognostic for STS patients (5). Importantly, co-expression of three stem
cell-associated genes, HIWI, hTERT and survivin, resulted in a significantly
increased risk of tumor-related death for STS patients no matter at which stage them
were (6). More recently, HIWI mRNA and protein expression was measured in ductal
adenocarcinoma of the pancreas (PDAC). Male patients showed a significantly
increased risk for tumor-related death in case of down- or upregulated expression of
HIWI mRNA (7).
Previously, we generated an anti-HIWI monoclonal antibody and demonstrated HIWI
expression rate being increased along with the development of gastric tumor from
precancerous tissues. Furthermore, the suppression of HIWI inhibited the growth of
gastric cancer cells and induced cell cycle arrest at G2/M phase, indicating HIWI was
associated with the proliferation of cancer cells (8). Recently, with same antibody, we
revealed that HIWI expression in esophageal tumor cells was closely correlated with
higher histological grade, clinical stage and poor prognosis (9). All these data suggest
that HIWI may play a pivotal role in tumor development.
To date, only few studies focused on HIWI protein expression, mainly due to the lack
of effective monoclonal antibody. In the present work, we further characterized 3
anti-HIWI monoclonal antibodies and examined the HIWI protein expression profile
in several cancer cell lines. HIWI expression in primary colorectal cancer tissue,
matched adjacent non-cancerous normal colorectal tissues, enteritis or colon adenoma
were also investigated by immunohistochemistry. The prognostic value of HIWI
protein for survival was also analyzed. We found an increased expression rate of
HIWI protein in colorectal cancerous tissue compared to adjacent non-cancerous
tissue and precancerous disease. Survival analysis revealed that HIWI positive
expression indicated a worse prognosis for early stage or lymph node metastasis
negative patients. Multivariate analysis demonstrated that HIWI over-expression was
a significant prognostic factor for overall survival with an increased risk. These
results suggested that HIWI may involve in certain events of colorectal cancer
development, such as metastasis, and could be a potential prognostic biomarker.
Patients and Methods
Patients and specimens
The study was conducted in accordance with the Declaration of Helsinki and was
approved and supervised by the Medical Research Ethics Committee of Peking
University Cancer Hospital & Institute. Informed consent was obtained from all of the
patients. Patients were followed up annually with a median follow-up time of 33
months (3 to 76 months). Histological classification and clinic pathological staging
were performed according to the TNM classification of UICC. A total of 270 patients
who underwent surgical resection of primary colorectal cancer between January 1999
and December 2002 in Beijing Cancer Hospital & Institute were investigated. The
patients didn’t receive any preoperative treatment such as chemotherapy, ethanol
injection or transarterial chemoembolization. Postoperative adjuvant chemotherapy
with 5-FU, leucovorin, and oxaliplatin combination (FOLFOX) or oxaliplatin and
capecitabine combination (XELOX) was given to all stage III/IV patients but not to
stage I/II patients. Formalin-fixed and paraffin-embedded specimens from these
patients and 236 matched adjacent non-cancerous normal colorectal tissues were
collected. 44 enteritis or colon adenoma tissues were also examined.
Generation of anti-HIWI monoclonal antibodies
Monoclonal antibodies against HIWI were generated by immunizing the Balb/C mice
with GST-HIWI fusion protein (321 amino acids of the N-terminal) emulsified in
Freund’s adjuvant (Sigma, St. Louis, MO, USA) (8). The spleen cells from the
immunized mice were fused with SP2/0 cells and the hybridoma clones were screened
with GST-HIWI (GST was used as the control). Three monoclonal antibodies (clone
2C5, 2G10 and 4D11) were obtained.
Cancer cell lines
Following cell lines were screened for HIWI protein expression: immortalized human
gastric mucosa epithelial cells GES-1; gastric cancer cells MGC803, SGC7901,
BGC823, AGS, N87, and MKN45; colorectal cancer cells LoVo, CL187, HT29, RKO,
SW480 and HCT116; hepatocellular carcinoma cells HepG2, BEL7402 and
SMMC7721; esophageal cancer cells E30, E70, E140, E180, E410, E450, E510,
YES2 and T12; lung cancer cells PG, GLC82, H446, H460, H1299 and A549. All the
esophageal cancer cells were kindly provided by Dr. Zhihua Liu of Cancer Institute,
Chinese Academy of Medical Sciences. MGC803, BGC823, BEL 7402 and
SMMC7901 cells were kept in our lab. Other cell lines were from ATCC (American
Type Culture Collection).
ELISA assay
The reactivity of mAbs with GST-HIWI and GST fusion protein was verified by
ELISA aasay. Ninety-six well plates were coated with 5g/ml GST or GST-HIWI
fusion protein in 0.1 mol/L biocarbonate buffer (pH9.5), then blocked with 5%
non-fat milk in PBS. Culture supernatants of three hybridomas were added to each
well and incubated for 1 h at room temperature, followed by washing five times with
PBS containing 0.1% Tween-20 (PBST). Bound mAb was detected by incubation
with HRP-conjugated anti-mouse IgG antibody (Zhongshan Co., Beijing, China) for 1
h, then by washing and the addition of 100 l o-phenylenediamine (0.4 mg/ml) in
citrate-phosphate buffer (pH 5.0), containing 0.02% (v/v) H2O2. The reaction was
stopped with 50 l of 12.5% H2SO4. OD492 was recorded by using a microplate
reader (Bio-Rad model 550).
Western blot
Cells were homogenized in lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L
NaCl, 1% NP-40, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 1 x
protease cocktail (Roche, Basel, Switzerland) for 20 min at 4 oC. The supernatant was
collected after centrifugation at 12,000 × g for 20 min at 4 oC and subjected to
Western blot with 2G10 monoclonal antibody. Anti-GAPDH antibody was used for
normalizing protein loading.
Immunohistochemistry
For immunohistochemical studies, 5 µm paraffin embedded sections were first placed
in an oven at 60 oC for 30 min prior to being deparaffinized and rehydrated by
sequential immersion in xylene and graded alcohol solutions. Endogenous peroxidase
activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 15
min. For antigen retrieval, slides were boiled in EDTA (1mmol/L; pH 8.0) for 10 min
in microwave oven and allowed to cool down for 45 min at room temperature.
Subsequently, slides were then blocked with 5% skim milk for 60 min, followed by
incubation with the anti-HIWI antibody 2G10 overnight at 4°C. EnVision+TM (DAKO,
Carpinteria, CA, USA) was used as the secondary antibody according to the
manufacturer’s instruction. Washes in PBS followed all steps. For the negative
controls, biotinylated normal goat IgG was substituted for the anti-HIWI antibody.
Antibody binding was visualized by a standard streptavidin immunoperoxidase
reaction, followed by chromagen detection with diaminobenzidine for 10 min and
hematoxylin counterstaining. The staining in the cytoplasm and the cytoplasmic
membrane was evaluated. The sections were photographed with a Spot RT color
camera coupled to a Nikon microscope. The score for HIWI staining was graded as
follow: no staining or staining observed in less than 10% of tumor cells was
considered negative; staining detected in more than 10% of tumor cells was
considered as positive. The immunostaining was evaluated independently by three
oncology pathologists without any knowledge of the clinical data.
Statistical analysis
χ2 tests were conducted to determine the association between HIWI expression and
the other variables. Survival curves were estimated using the Kaplan–Meier method
and compared by the log rank test. Overall survival was defined as the time from the
date of histological diagnosis to the date of last contact or death from any cause.
Analogously, recurrence free survival was the time to first diagnosis of recurrence or
to the last follow-up visit establishing a tumor free state. A multivariate analysis was
performed by using the Cox regression model (a backward selection) to assess
whether a factor was an independent predictor of DFS. Hazard ratios (HRs) with 95%
confidence intervals were estimated. A two-tailed P value of less than 0.05 was
considered statistically significant. All statistical analyses were performed with SPSS
v13.0 software (SPSS Inc., Chicago, IL, USA).
Results
Generation, characterization of novel anti-HIWI monoclonal antibodies and
HIWI expression profile in diverse cancer cell lines
To generate antibodies with high specificity and sensitivity for HIWI protein, Balb/C
mice were immunized with GST-HIWI protein. By fusing spleen cells with SP2/0
myeloma cells, 900 surviving hybridoma clones were isolated and grown up.
Supernatants from each clone of hybridoma cells were screened using ELISA. Finally,
3 hybridomas that specifically recognized GST-HIWI, but had no reaction with GST
were established (Figure 1A). Next, we tested their applications in other
immunoassays. All these antibodies recognized GST-HIWI in the Western blot
analysis (data not shown).With protein lysates from SW480 colorectal cancer cells,
we found that clone 2G10 was superior to clone 2C5 or 4D11 in recognizing
endogenous HIWI protein in Western blot analysis (Figure 1B), whereas 2C5
antibody was ideal for FACS analysis (Fluorescence Activated Cell Sorting) (data not
shown). Next we used 2G10 antibody to examine HIWI expression in a panel of
cancer cell lines originated from diverse human tissues, including stomach, colorectal
tract, lung, liver and esophagus. Results revealed varying levels of HIWI in these cell
lines (Figure 1C).
HIWI expression in colorectal tissues
In Western blot, we found the high level of HIWI protein expression in all of six
colorectal cancer cell lines (Figure 1C). We then investigated HIWI expression in
colorectal cancer tissues and matched adjacent non-cancerous tissues by
immunohistochemical analysis with 2G10 antibody. HIWI was negative in enteritis
tissue (Figure 2A) and most of adjacent non-cancerous colorectal tissues (Figure 2B).
Expression of HIWI in cancerous tissue was revealed in 69 out of 270 cases (25.6%).
HIWI was mainly detected in the cytoplasm of the colorectal carcinoma cells (Figure
2C). Interestingly, there were 15 HIWI positive cases in 236 (6.4%) matched adjacent
non-cancerous tissues (Figure 2D). No HIWI expression was observed in colon
adenoma tissue in the tested samples (data not shown).
Prognostic value of HIWI expression for colorectal cance patients
Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that
patients with HIWI positive expression in colorectal cancer tissue had lower overall
survival (OS: 39.3% versus 55.0%) and disease free survival (DFS: 40.1% versus
54.5%) compared with negative patients, though the difference didn’t reached
statistical significance (Figure 3A). However, patients with positive HIWI expression
in adjacent non-cancerous tissue had statistically lower OS and DFS compared with
negative patients: OS: 10.4% versus 55.5% P=0.009, DFS: 10.4% versus 55.1% P=
0.015 (Figure 3B). Further stratum analysis was performed according to
clinicopathological variables of patients including clinical TNM stage and lymph
node metastasis. Patients were stratified into early stage group (phase I and II, n=134)
or late stage group (phase III and IV, n=136) and lymph node positive or negative
group. In early stage group, patients with positive HIWI expression in colorectal
cancer tissue (n=27) had significantly lower overall survival rate (OS: 57.4% versus
79.5% P=0.014) and disease free survival rate (DFS: 56.7% versus 80.5% P= 0.010)
compared with negative patients (Figure 3C). It is the same in lymph node negative
group (n=147) that patients with positive HIWI expression in colorectal cancer tissue
(n=35) have significant lower overall survival rate (OS: 53.0% versus 73.5% P=0.037)
and disease free survival rate (DFS: 52.2% versus 74.6% P= 0.025) compared with
HIWI negative patients (Figure 3D).
Multivariate analysis demonstrated that HIWI expression is an independent
prognostic factor for patients
Further multivariate analysis also demonstrated that HIWI over-expression was an
independent significant prognostic factor for overall survival with an increased risk of
1.675 (95%CI：1.132-2.479，P=0.010) for patients with positive as compared to
negative. Besides HIWI expression, relapse, TNM stage and intravascular embolism
are all prognostic factors (Table.1).
Discussion
Cancer stem cells refer to a subset of cancer cells, which self-renew to generate
additional cancer stem cells and differentiate to generate phenotypically diverse
cancer cells with limited proliferative potential (10, 11). Further characterization of
cancer stem cells-related factors might lead to improved diagnostics and therapies by
allowing us to better understand the development of cancer (12, 13). HIWI is a human
homologue of PIWI gene, being responsible for stem cell self-renewal, RNA interfere
and translational regulation (1, 2). Several studies, including work in our laboratory,
have demonstrated the expression of HIWI in a variety of tumors (5-9). Previously we
demonstrated that silencing of HIWI inhibited proliferation of AGS gastric cancer
cells (8). Recently, HIWI expression was found to be positively correlated with
angiopoietin-2 in uterine cervical cancer, breast carcinoma and ovarian cancer,
indicating HIWI may play a role in cancer angiogenesis (14).
Here, we report the expression profile of HIWI protein in a panel of cancer cell lines
and in colorectal cancer tissues by using novel anti-HIWI monoclonal antibody. HIWI
protein exhibited varied level of expression in cell lines established from different
tissues. HIWI levels were detected in different esophageal cancer cells, which were
complementary to the results we reported in esophageal cancer tissues (9). Positive
rate of HIWI in primary colorectal cancer tissue was significant higher than matched
adjacent non-cancerous normal colorectal tissues, enteritis or colon adenoma.
Recently, HIWI protein expression was detected in pancreas adenocarcinoma in a
small sample size by immmunostaining using a polyclonal antibody and the positive
staining was detected in 21 out of 78 patients (26.9%), which is quite similar with
what we found in colorectal carcinoma (7). Colorectal cancer patients with positive
HIWI protein showed a tendency of worse outcome, though there was no statistical
significance. However, multivariate analysis demonstrated that HIWI over-expression
was a statistically significant prognostic factor for overall survival. Similarly, elevated
or reduced HIWI mRNA level were significantly associated with a poor outcome for
soft-tissue sarcoma patients (5). Furthermore, in early stage or no lymph node
metastasis patients, HIWI protein showed a strong prognostic value in both OS and
DFS, suggesting HIWI as a stem cell-associated gene may have impact on early stage
of tumorigenesis. As in leukemia, HIWI gene is exclusively expressed in CD34+
haematopoietic progenitor cells but not in more differentiated cell populations (3).
Because of HIWI’s putative contribution to the development of cancer in the early
stage, we were prompted to perform serological analysis of HIWI by developing
Sandwich ELISA method, however, no HIWI protein was detected in the sera from
colon cancer patients or healthy controls (data not shown).
Although HIWI was found to be overexpressed in diverse types of cancers, signals
and pathways dictating HIWI expression remain to be determined. The natural
pentacyclic triterpene betulinic acid was reported to perturb cell cycle progression and
inhibit proliferation of AGS gastric cancer cells, in part by down-regulating HIWI and
cyclin B1 expression (15). Unlike HIWI, regulation of PIWI had been extensively
studied. Genetic interaction between the PIWI signaling network and Hedgehog
signaling pathway may regulate the division of both germ line stem cells and somatic
stem cells (16). Hedgehog pathway is critical for developmental patterning system
and the development of cancers by modulating cell cycle progression through diverse
interacting proteins (17, 18), therefore it will be interesting to investigate the physical
and functional interactions between PIWI/HIWI and Hedgehog-interacting proteins.
Besides PIWI, several PIWI/HIWI-like proteins had been identified, some of them
play essential roles in tumorigenesis through mechanisms not limited to controlling
RNA metabolism (19). For example, PL2L (PIWI-like2-like) promotes tumorigenesis
by enhancing expression of bcl2 and stat3 gene and the nuclear localization of NF-κB
(20). Whether HIWI has similar effect will be explored in the future study.
In conclusion, we report for the first time the increased expression of HIWI in
colorectal cancer. Positive staining in colorectal cancer tissue or matched adjacent
non-cancerous tissue is a poor prognostic factor for the colorectal cancer patients,
especially for those in early stage and those with no lymph node metastasis. Based on
these data, we propose that HIWI may have vital effect on colorectal cancer
development and could be a potential prognostic and therapeutic target for colorectal
cancer patients. However, more studies still should be done to elucidate the molecular
mechanism of HIWI in colorectal carcinogenesis.
Acknowledgement
We thank Dr. Jiyou Li (Peking Cancer Hospital & Institute) for providing colorectal
tissue samples and Dr. Zhihua Liu (Cancer Institute, Chinese Academy of Medical
Sciences) for kindly providing esophageal cancer cells lines.
Conflict of Interest Statement
Authors claim no conflict of interest.
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Figure Legends
Figure 1. Characterization of anti-HIWI antibodies and HIWI wexpression
profiles in diverse cell lines.
A. Characterization of the specificity of HIWI antibody by ELISA assay. Reactivity
of culture supernatant from three indicated hybridomas was tested for binding to
GST-HIWI and GST protein by ELISA. Bound antibodies were detected with
HRP-conjugated anti-mouse antibody. Pre-immune serum was used as negative
control. B. Characterization of the specificity of anti-HIWI mAbs against endogenous
HIWI protein. Equal amount of protein lysates from SW480 cells was resolved by
SDS-PAGE, followed by Western blot with indicated mAbs. Blots were subjected to
ECL with same exposure time to compare mAb’s specificity and sensitivity. Antibody
against GAPDH was used to normalize loading. C. HIWI expression in cancer cell
lines established from different tissues. Equal amount of protein lysates from
indicated cell lines was subjected to SDS-PAGE and Western blot analysis with 2G10
antibody.
Figure 2. Immunohistochemical staining of HIWI in malignant and benign
colorectal tissues with 2G10 antibody. A. Enteritis tissue, HIWI negative (200X). B.
Adjacent non-cancerous normal colorectal tissue, HIWI negative (200X). C.
Colorectal cancerous tissue, HIWI positive (200X). D. Colorectal cancerous tissue
and adjacent non-cancerous colorectal tissue, both were HIWI positive (200X).
Figure 3. Kaplan-meier estimates of overall survival and disease-free survival with
respect to HIWI expression and clinicopathological parameters. (A) OS and DFS time
stratified by HIWI expression in cancer tissues. (B) OS and DFS stratified by HIWI
expression in adjacent non-cancerous tissues. (C) OS and DFS for the subgroups of
early stage patients (stage I/II) stratified by HIWI expression. (D) OS and DFS for the
subgroups of patients with no lymph node metastasis stratified by HIWI expression.
Table 1. Multivariate Cox’s regression analysis: HIWI expression in
cancerous tissue is an independent prognostic factor.
95% CI, 95% confidence interval. Statistically significant (p < 0.05)
Variables
HIWI (+)
relapse (+)
TNM stage (III)
Intravascular
embolus (+)
Overall Survival
95% CI
P
relative
lower upper
risk
1.67
1.132 2.479 .010
5
2.564 5.717 .000
3.82
8
2.029 3.370 .000
2.61
4
1.261 2.708 .002
1.84
8