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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 5g/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. Reference 1. Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. 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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