Colorectal Cancer Lymph Node Metastasis Molecules Related To Quantitative Proteomics Research

AIM:Tumour metastasis remains one of the major challenges in management for colorectal cancer (CRC) patients. Lymph node metastasis (LNM), CRC migration through the lymphatic route and deposition tumor cells into local lymph nodes, is the most common metastasis form in CRC. The status of the local lymph nodes delivers crucial information concerning cancer staging, prognosis, and the decision of clinical management on the understanding that the existence of LNM notably reduces the chance of CRC survival. To investigate the molecular mechanisms related to LNM and to improve the diagnosis and prognosis of CRC, we employed a quantitative proteome analysis to profile the differently expressed proteins associated with LNM for CRC, which could present messages to the mechanisms of LNM, development of new biomarkers for CRC and decision of clinical personalized treatment.METHODS:Patients and samples:Totally,144 colorectal carcinoma samples were collected under informed consent in our hospital (Fudan University Shanghai Cancer Center, Shanghai, China). All the patients received neither chemotherapy nor radiotherapy before surgery. Resected specimens were reviewed by two senior pathologists according to the criteria described in the American Joint Committee on Cancer's (AJCC) Cancer Staging Manual (Six Edition,2002). For the screening and confirmation study,32 primary CRC tissue samples which underwent curative resection in 2009 were collected and divided into two groups of LNM CRC and non-LNM CRC with sixteen cases in each group. The number of lymph nodes retrieved was not less than 12 in non-LNM CRC. None of them had distant metastasis. The fresh colorectal tumor tissues were obtained immediately after the surgery. For the S100A4 expression study, paraffin-embedded tissues in another independent set of 112 primary CRC between January 2004 and August 2004 were used for immunohistochemistry assessment. Ethical approval was obtained from the Cancer Hospital Research Ethics Committee. Sample preparation, methyl esterification stable isotope labeling and 2D-LC-MS/MS (two-dimensional liquid chromatography followed by tandem mass spectrometry):Each frozen samples were extracted followed by concentration assessment. Equal amount of each sample from LNM CRC and non-LNM CRC groups was pooled together respectively. One hundred micrograms of proteins from each sample pool were digested with trypsin. The peptides from LNM CRC sample were tagged with d0-methanolic HC1, whereas those from non-LNM CRC sample were labeled with d3-methanolic HC1. The two resulting peptides were mixed. Then the pooled sample was separated by a two-dimensional microcapillary high-performance LC system, followed by MS/MS analysis using an LTQ Orbitrap.Data analysis:Bioworks 3.3.1 was used to generate the peaklists of all acquired MS/MS spectra, which were then automatically searched against a database of International Protein Index (IPI) human 3.43 using SEQUEST. Quantification of the ratio of each protein was achieved using the Xpress program. Proteins with expression fold change> 2.5, p< 0.05 were defined as differentially expressed proteins.Western blotting:Thirty micrograms of proteins from the each of the same samples as screening were used for western blotting.β-actin was detected simultaneously as a loading control. All blots were visualized using ECL detection system and quantitated by densitometry using a LAS-2800 imager. Immunohistochemistry:S100A4 expression and location distribution in cancer cells were examined immunohistochemically using paraffin-embedded tissues. Each sample was graded according to the intensity and extent of stainingReal-time quantitative PCR:Total tissue RNA was extracted and the Real-time quantitative PCR analysis was performed according to the manufacturer's instructions.β-actin was applied as an internal control. For relative quantification,2-△△Ct was calculated and used as an indication of the relative expression levels.RESULTS: Quantitative proteome analysis with methyl esterif ication stable isotope labeling combined with 2D-LC-MS/MS:As a result, the quantitative differential expression of 644 proteins was identified, after calibration ofβ-casein. Significantly,43 of these (6.7%) proteins were displayed differentially (at least 2.5-fold) in LNM CRC compared with non-LNM CRC. Among these 43 proteins,28 were found to be up-regulated in LNM CRC, and 15 were down-regulated. These proteins were divided according to the molecular function employing the tools at www. geneontology. org, Which were divided into nine groups:enzyme activity, binding activity, motor activity, signal transduction, transcription regulation, enzyme regulation, transporter activity, apoptosis regulator activity, and others.Confirmation of S100A4 expression by western blotting, immunohistochemistry and real-time quantitative PCR:In the study of western blotting, the expression of S100A4 was dramatically higher in LNM CRC compared with non-LNM CRC (p<0.001). immunohistochemically, there was no immunoreactivity in normal mucosa, weak staining in cancer cells of non-LNM group, and strong staining in cancer cells of LNM group and marched metastatic lymph node. Positive staining is present mainly in the cytoplasm and/or nucleus of cancer cells. Real-time quantitative PCR revealed that S100A4 mRNA level was higher in LNM CRC than in non-LNM CRC, which is consistent with the trend at protein level(p< 0.001). Association of S100A4 expression with clinicopathological features and postoperative prognosis of CRC patients:After division of these patients into S100A4-positive and S100A4-negative groups, statistical analysis revealed that positive expression of S100A4 significantly associated with lymph node metastases, and advanced TNM stage (p< 0.001). However, no significant correlations were observed between S100A4 expression and other clinicopathological parameters of gender, age, tumour size, tumour differentiation and tumour location. Furthermore, we found that the 5-year cumulative recurrence rate was significantly higher in patients with S100A4-positive CRC than in S100A4-negative group(p< 0.001) and the 5-year cumulative survival rate in patients with S100A4-positive CRC was much lower than in patients with S100A4-negative CRC(p< 0.001). Univariate analyses and multivariate analysis indicated S100A4 expression was an independent prognostic factor of recurrence and overall survival (p< 0.05).CONCLUSION:our current study employed a quantitative proteome analysis to profile the differently expressed proteins associated with LNM for CRC. S100A4 was identified and confirmed to be significantly over expressed in LNM CRC. Further evaluation in a large-scale samples suggested that S100A4 might act as a powerful biomarker for LNM and prognosis in CRC. We also identified a number of proteins besides S100A4, which might provide a more profound insight into the mechanism of LNM in CRC and deserve deeper research.