Lectin from Agrocybe aegerita as a Glycophenotype Probe for Evaluation of Progression and Survival in Colorectal Cancer

Colorectal cancer (CRC) is a major health problem worldwide, with global increases in incidence and death due to an expanding and aging population. Based on the World Health Organization cancer statistics in 2013, there is over 1.36 million new cancer cases and 694,000 deaths to have occurred (Ferlay et al., 2013). Exceptional amount of high-throughput experiments have been performed for well validated prognostic markers (Balog et al., 2012). However, the clinical useful markers based on genomics and proteomics remain limited. In recent years, aberrant glycosylation has been recognized as a cellular hallmark event during carcinogenesis (Reis et al., 2010; Adamczyk et al., 2011), and it has been a topic for well characterization of tumor and better understanding the mechanism (Meany et al., 2011). Both Nand O-linked glycoproteins have been shown to be altered in a wide


Introduction
Colorectal cancer (CRC) is a major health problem worldwide, with global increases in incidence and death due to an expanding and aging population.Based on the World Health Organization cancer statistics in 2013, there is over 1.36 million new cancer cases and 694,000 deaths to have occurred (Ferlay et al., 2013).
Exceptional amount of high-throughput experiments have been performed for well validated prognostic markers (Balog et al., 2012).However, the clinical useful markers based on genomics and proteomics remain limited.In recent years, aberrant glycosylation has been recognized as a cellular hallmark event during carcinogenesis (Reis et al., 2010;Adamczyk et al., 2011), and it has been a topic for well characterization of tumor and better understanding the mechanism (Meany et al., 2011).Both N-and O-linked glycoproteins have been shown to be altered in a wide
Lectins are proteins or glycoproteins that can recognize specific glycan structures.To investigate the significance of distinct glycosylation states, it is crucial to identify new lectins that discriminate unique sugar structures among kinds of oligosaccharides.AAL (Agrocybe aegerita lectin) from the edible mushroom A. aegerita is an antitumor protein that exerts its tumor-suppressing function via apoptosis-inducing activity in cancer cells and animal models (Zhao et al., 2003).We demonstrate that AAL belongs to galectin family with a unique carbohydrate recognition domain that specifically recognizes β-galactose (Liang et al., 2009).The recent glycan-array analysis reports that glycans with high affinity to AAL contain [3OSO3]Galβ1-3GalNAcα-Sp8 (sulfated TF disaccharide fixed to a spacer arm Sp8-CH2CH2CH2NH2), [3OSO3] Galβ1-3GalNAcα-Sp8, Neu5Acα2-3Galβ1-3GalNAcα-Sp8 (Sialyl-TF disaccharide) and Galβ1-3GalNAcα-Sp14 (TF disaccharide) etc (Feng et al., 2010).The observations lead to this assessment of using AAL for understanding the glycosylation changes associated with the development of colorectal cancer.

Patients and tissue samples
CRC and corresponding non-tumor colorectal tissues were obtained from 59 patients as described previously (Huang et al., 2012).All samples were graded histopathologically by two experienced pathologists.Tumor staging was established according to 7th edition of the Cancer Staging Manual of the American Joint Committee on Cancer (AJCC) (Gunderson et al., 2010).Clinical data related to the samples used in this study, including age, gender, tumor stage, and original tumor site were summarized in Table 1.The patients were followed up annually with a median follow-up time of 70 months.Informed consent was obtained from all subjects, and this study was approved by the Institutional Ethics Committee.

Lectin-histochemistry detection
Purified AAL with a His6 tag was biotinylated performed by Beijing Biosynthesis Biotechnology CO.LTD.All paraffin-embedded tissues were deparaffinized in xylene and rehydrated in a graded ethanol series according to routine histochemistry.The sections were boiled in EDTA (1 mM; pH8.0) for 15 mins in a microwave oven for antigen retrieval, and cooled down in phosphate-buffered saline (pBS), pH 7.2 at room temperature.Endogenous peroxidase activity was eliminated using 0.3% (v/v) H 2 O 2 in methanol for 30 mins.The non-specific glycoconjugate binding was blocked using 0.5% (v/v) periodate treated BSA in PBS for 30 mins at room temperature.Sections were incubated with biotinylated AAL 20 µg/ml at 37℃ for 1 h and with 12.5 µg/ml horseradish peroxidaseconjugated streptavidin (proteintech group) for 40 mins.After additional washes, bound lectin was visualized with diaminobenzidine tetrahydrochloride (DAB), and then specimens were counterstained with hematoxylin.A positive control and a negative control (without lectin) were included in each run.

Statistical analysis
Statistical analysis was performed using the SPSS software package (version 16.0).The chi-square test or Fisher's extract test were used to analyze the correlations between clinicopathological features and AAL histochemistry score of CRC patients.Survival analysis was performed using the Kaplan-Meier method with the log-rank test and Breslow test.The prognostic significance of each clinicopathological feature was determined using the univariate and multivariate Cox regression analyses.In a multivariate Cox proportional hazard model, the independent prognostic factors were identified from the significant predictors in univariate analysis with an enter mode.

AAL specific glycoconjugate expression and clinicopthological associations
The glycoconjugate expression recognized by AAL in the colorectal cancer tissues and corresponding adjacent normal counterparts were examined using immunohistochemistry staining.The representative results are shown in Figure 1.Various levels of immunoreactivity for AAL were found in cancer and adjacent non-tumor regions.The AAL specific glycoconjugate expression was significantly higher in tumor tissues than corresponding normal tissues (66.1% and 46.1%, respectively, p=0.037).The associations between lectin-histochemistry score and clinicopathological parameters were assessed using chisquare test or Fisher's exact test.The results indicated that AAL specific glycoconjugate expression was correlated with depth of invasion (p=0.015) and TNM stage (p=0.024,Table 1).

Associations between AAL specific glycoconjugate expression and patient survival
Kaplan-Meier analysis with the log-rank and Breslow test were performed to analyze the correlation between AAL specific glycoconjugate expression and patient survival.According to the Kaplan-Meier survival curves, the higher expression of AAL specific glycoconjugate is a significant prognostic factor for poor overall survival and progress free survival in CRC patients.Patients with lower AAL specific glycoconjugate expression levels have a significantly higher survival rate than that with higher expression (78.9% for lower expression group and 50.0%for higher expression group, respectively, p=0.046 by log rank test and p=0.047 by Breslow test for overall survival, Figure 2A; 75% for lower expression group and 50% for higher expression group, respectively, p=0.054 by log rank test and p=0.038 by Breslow test for progress free survival, Figure 2B).
To identify the AAL specific glycoconjugate expression and other variables of potential prognostic significance in all of the patients with CRC, univariate and multivariate Cox regression models were performed.However, only a marginally significant association was found between AAL specific glycoconjugate expression and overall survival or progress free survival by Univariate Cox regression analysis (p=0.059 for overall survival, Table 2; and p=0.064 for progress free survival, Table 3).The univariate Cox regression analysis indicated that clinical variables including lymph node status, metastasis and pathological stage were significantly associated with overall survival (p=0.001,p=0.002, and p=0.001, respectively, Table 2) and with progress free survival (p=0.001,p=0.004, and p=0.002, respectively, Table 3).Furthermore, multivariate Cox regression analyses were performed to evaluate the potential of AAL specific glycoconjugate expression as an independent predictor for overall survival or progress free survival of CRC.The results showed that no independent predictor was found with or without AAL's addition.
Glycoconjugate markers for colon cancer include aberrant mucins, cadherins, selectins and Ig-like adhesion molecules, glycoconjugate components of ECM, etc (Szajda et al., 2008).Expression of TF antigen on glycoconjugates of the cell surface has been correlated with tumor prognosis and metastasis (Baldus et al., 2000;Szajda et al., 2008).Lately, sulfated and sialylated glycans have been detected to be increased in tumor tissues or plasma samples from colorectal cancer patients (Qiu et al., 2008;Balog et al., 2012).High level of sialylation has been observed on core 1 type glycan (TF structure) from breast cancer patient (Storr et al., 2008) and colorectal cancer patient (Schneider et al., 2001).According to sulfated glycoconjugates, only few studies have correlated the increase in sulfated glycans with cancer in general.Gal-3-O-sulfotransferase is expressed in most breast cancer cells and colon cancer cells, which is specific for T-hapten Galβ1-->3GalNAcα-and the Galβ1-4GlcNAc terminal unit in O-glycans respectively (Chandrasekaran et al., 2006).These results suggest that sialic acid and sulfated glycoconjugates, especially the sialyl-TF and sulfated TF may act as potential tumor-related biomarker in cancer.Some of our recent findings show that AAL specifically recognizes the sulfo-TF antigen expressed on malignant human hematopoietic cells resulting in apoptosis with the activation of caspase-8, -9, and -3 (data not published).The present study finds the significant relationship between prognosis and staining characteristics for AAL in colorectal cancer tissues (Figure 1 and 2), which suggests that sialoglycoconjugates and sulfoglycoconjugates recognized by AAL are related to worse prognosis for colorectal cancer patients.Multivariate Cox regression analyses have not found the potential of AAL specific glycoconjugate expression as an independent predictor, but there is a marginally significant association between its expression and overall survival or progress free survival by Univariate Cox regression analysis (Table 2 and 3), which may due to the limited amount of tumor tissues.
Although the study has discussed the importance of aberrant glycoconjugats histochemically detected by AAL, the nature of these glycoconjugates remains unknown.Carbohydrate moieties of glycoconjugates are constructed by complex interaction involving a series of glycosyltransferases (Essentials of Glycobiology, 2009).Differences in AAL staining intensity might be due to quantitative differences in the expression level of glycosyltransferases. Enzyme activity of alpha2,3-sialyltransferase (ST3Gal-I) has been found to be significantly increased in carcinoma specimens compared with normal mucosa (Schneider et al., 2001), and Gal-3-Osulfotransferase with preference for the Galβ1-4GlcNAc terminal unit in O-glycans is expressed in colon cancer cells (Chandrasekaran et al., 2006).These finding is consistent to the levels of sialoglycoconjugates and sulfoglycoconjugates expression recognized with AAL.
There have been specific monoclonal antibodies recognizing sialyl TF glycan antigens (Essentials of Glycobiology, 2009), but to our knowledge, no lectin or antibodies have been found to bind sulfo TF.Sulfation of TF has been reported to lead to modulation of galectin-1 interaction with glycoconjugate and play a role in galectin functions (Allen et al., 1998).SB1a with terminal sulfo TF disaccharide on the cell surface of human colon adenocarcinoma cells has been reported to be the important ligand for galectin-4 and promote cell adhesion (Ideo et al., 2002;Ideo et al., 2005).AAL, with high affinity with sulfo TF, could be a useful tool for the investigation of the glycan.
The prognostic information provided by AAL lectin histochemistry may be used clinically to inform the physician and aid treatment decisions; far more interesting is the challenge of further understanding the precise nature of the AAL-binding ligands, and defining their role in the progress of the disease.

Table 3 . Univariate and Multivariate Analysis of Individual Parameters for Correlations with Progress Free Survival Rate: Cox Proportional Hazards Model
*p<0.05