Screening And Preliminary Identification Of Molecular Signature Of Metastasis In Colorectal Cancer

BACKGROUND & OBJECTIVEMetastasis is the main cause leading to deaths of patients with colorectal cancer. Micrometastasis,which is present in more than half of patients with colorectal cancer before radical surgery,resulted in distant metastasis and recurrence in colorectal cancer after radical surgery.However,there is no reliable method to detect potential micrometastases in patients with colorectal cancer.Therefore,to clarify the molecular mechanism of metastasis in colorectal cancer is the main task in the field of colorectal cancer research.Molecular signature is a whole number of some genes,to determine or define certain biological characteristics.Different with individual molecules,molecular signature pay more attention to the coordination of different genes,it is the overall description of different biological characteristics and it is based on function research of a large number of single gene.An important means of gaining molecular signature is through Analysis of gene expression profiles.In this study,59 single-cell-derived progeny(SCPs) cell subclones of colorectal cancer were established in vitro and selected in in surgical orthotopic implantation mouse model of colorectal cancer.Several SCPs subclones with different metastatic potential were obtained in our study.We identified genes associated with tumor metastasis by comparing the difference between subclones with different metastatic potential and its parent cell line using genome-wide expression profile chip of Affymetrix.Molecular signature of metastasis in colorectal cancer was extracted from metastasis associated genes based on bioinformatic methods and literature mining methods.The clinical significance of molecular signature and the comprising genes was tested in clinical samples of colorectal cancer.METHODS1,Single cell-derived progenies(SCPs) of colorectal cancer were isolated from SW480/EGFP cells.Tumor formation of SCPs was detected by monitoring the tumor growth under subcutaneous tissue of nude mice.In vitro methods and orthotopic implantation mouse model of colorectal cancer were used to screen for SCPs with different metastasis potential2,Gene expression profiles of SCPs with different metastasis potential and its parent SW480/EGFP were determined using Affymetrix human Genome U133 Plus 2.0 Array containing 47000 transcripts.Hybridization,washing,staining, scanning,and data collection were performed according to the standard Affymetrix protocol.Gene sets shared in two independent comparision between SCPs and its parent SW480/EGFP were selected in our next study.Molecular signature of metastasis in colorectal cancer was extracted from metastasis associated genes based on bioinformatic methods and literature mining methods.3,Biological functions,pathways and the possible role in tumor metastasis of genes in our molecular signature of metastasis were analyzed.4,Formalin-fixed and paraffin-embedded tissues of CRC were used in our clinicopathological investigation.Immunohistochemistry was performed using a Dako Envision System(Dako,Carpentaria,CA,USA) following the manufacturer's recommended protocol to study protein expression of five genes in our metastasis signature.Antibodies against LYN,SDCBP,MAP4K4,MID1,DKK1 were used in this study.The clinical significance of molecular signature and the comprising genes was tested in clinical samples of colorectal cancer.5,All statistical analyses were carried out using the SPSS software program (version 12.0;SPSS Inc.,Chicago,IL).The chi-square test was used to assess differences in metastases and peritoneal dissemination of SCPs and their parental SW480/EGFP.The Mann-Whitney U test was used to analyze the relationship between expression of LYN,SDCBP,MAP4K4,MID1,DKK1 and clinicopathological characteristics of the patients.Survival curves for the patients with different expressions of LYN,SDCBP,MAP4K4,MID1,DKK1 were plotted using the Kaplan-Meier method and compared using the log-rank test.The significance of various survival-related variables was assessed using the Cox proportional hazards model in a multivariate analysis.A p-value of≤0.05 will be considered statistically significant.RESULTS1,Fifty-nine single cell-derived progenies(SCPs) from SW480/EGFP was isolated and established by limited dilution method.2,Tumor formation under subcutaneous tissue of nude mice was found in 29 SCPs.Heterogeneous ability of tumor formation was found in different SCPs from SW480/EGFP(F=33.446,P=0.000).3,Surgical orthotopic implantation(SOI) nude mice model of colorectal cancer was performed to compare metastatic potentials of 29 SCPs and their parent SW480/EGFP.Different metastatic potentials were found in 29 SCPs in SOI nude mice model of colorectal cancer(F=4.155,P=0.000).Among the SCPs studied,3 SCPs with high metastatic potential isolated as SCP12,SCP21 and SCP51,3 SCPs with low metastatic potential isolated as SCP11,SCP28 and SCP58,were found in SOI nude mice model of colorectal cancer.In order to rule out the randomness of tests,two SCPs with high metastatic potential and 2 SCPs with low metastatic potential were tested repeatedly to confirm their metastatic potential in SOI nude mice model of colorectal cancer.Statistically significant differences of metastasis in different SCPs was observed(F=6.172, P=0.029).A SNK multiple comparison showed that SCP51 had the highest metastatic potential and SCP58 had the lowest metastatic potential among the above SCPs.SCP51 and SCP58 were used in our next study.4,Gene expression profiles of SCP51,SCP58 and its parent SW480/EGFP were detected by HG-U133 plus 2.0 oligonucleotide array of Affymetrix Inc.SAM analysis was performed to identify the most discriminating genes.One hundred and forty-three genes including 85 up-regulated genes and 58 down-regulated genes were extracted as gene expression pattern of metastasis in colorectal cancer;Hierarchical cluster analysis showed that the cell subclone and its parent cell line can be successfully classified as three categories according to gene expression level of 143 genes.5,Molecular signature of metastasis in colorectal cancer comprising of 29 genes was extracted using platform of Significance Analysis of Microarrays constructed by Capitalbio company.6,Biological significance of genes in the molecular signature of metastasis in colorectal cancer was analyzed by literature mining method.Five genes including LYN,SDCBP,MAP4K4,MID1 and DKK1 were found to be associated with tumor metastasis.However,no study about the roles of five genes was found.7,We found that mRNA expressions of LYN,SDCBP,MAP4K4,MID1,DKK1 in SCP51,SCP58 and SW480/EGFP cells detected by real-Time PCR were the same as the results detected by previous gene chip.We found that mRNA expression levels of LYN,SDCBP,MAP4K4 were higher in the primary colorectal tumor tissue samples than those in their normal counterparts.We also observed that mRNA expression levels of DKK1 and MID1 were lower in the primary colorectal tumor tissue samples than those in their normal counterparts.8,Immunohistochemistry results showed that LYN,MAP4K4 and MID1 were closely correlated with metastasis of patients(P＜0.05) and LYN,SDCBP, MAP4K4,MID1 were closely associated with the prognosis of patients(P＜0.05).9,To determine the possibility of using LYN,SDCBP,MAP4K4,MID1 and DKK1 as a molecular signature in colorectal cancer,the sum of 5 independent scores of LYN,SDCBP,MAP4K4,MID1 and DKK1 in clinical samples of colorectal cancer was used as the final score of the molecular signature in colorectal cancer.The results showed the molecular signature comprising of LYN,SDCBP,MAP4K4,MID1 and DKK1 was significantly correlated with metastasis and prognosis of patients(P＜0.05) as a whole.Our results suggested that the molecular signature comprising of LYN,SDCBP,MAP4K4,MID1 and DKK1 can be potential new molecular markers of metastasis and prognosis in colorectal cancer.CONCLUSIONS1,SW480/EGFP exhibiting heterogeneity in metastatic potential is suitable for in vivo selection and SCPs selection. 2,Different SCPs from SW480/EGFP exhibit heterogeneity in metastatic potential and tumorgenicity detected in vitro assay,in vivo tumor growth and metastasis assays.3,One hundred and forty-three genes including 85 up-regulated genes and 58 down-regulated genes were extracted as gene expression pattern of metastasis in colorectal cancer,Molecular signature of metastasis in colorectal cancer comprising of 29 genes was determined using bioinformatic methods and text mining.4,LYN,MAP4K4 and MID1 were closely correlated with metastasis of colorectal cancer and LYN,SDCBP,MAP4K4,MID1 were closely associated with the prognosis of colorectal cancer.Molecular signature comprising of LYN,SDCBP, MAP4K4,MID1 and DKK1 was significantly correlated with metastasis and prognosis of patients and can be potential new molecular markers of metastasis and prognosis in colorectal cancer.INNOVATIONS1,Fifty-nine single cell-derived progenies(SCPs) from SW480/EGFP is isolated and established.2,SCPs with high metastatic potential isolated as SCP12,SCP21 and SCP51,SCPs with low metastatic potential isolated as SCP11,SCP28 and SCP58,are established and confirmed in SOI nude mice model of colorectal cancer.3,Gene expression pattern and molecular signature of metastasis in colorectal cancer have been determined using bioinformatic methods and text mining.4,LYN,SDCBP,MAP4K4,MID1 are the candidates of prognostic indexes of colorectal cancer.Molecular signature comprising of LYN,SDCBP,MAP4K4, MID1 and DKK1 can be potential new molecular markers of metastasis and prognosis in colorectal cancer.