Study On The Correlation Of EIF3e In Colon Cancer

BackgroundMalignant tumor is the world’s largest lethal disease, it sriously threatens human’s life and health, and it may affect any part of the human being More than 100 kinds of malignent human tumors have been found in the world so far, the five most commen malignent tumors in men are lung, liver, colorectal and esophageal cancer; while in women are breast, lung, gastric, colorectal and cervical cancer. Along with the changing of living habits and the diet structure, and the acceleraction of the aging process, the morbidity and mortality of colorectal cancer(CRC) in our country are ranked fifth and third respectively in male and femal malignent tumors, and are becoming higher and younger.Colon cancer arises through a multistep process in which genetic and epigenetic aberrations accumulate in a sequential order to drive malignant transformation of normal colon cells. Some of these aberrations, for instance, microsatellite instability, BRAF mutation,18q loss of heterozygosity, KRAS, p53, guanylyl cyclase 2, ERCC-1, had been validated as biomarkers for predicting chemoresistance and prognostic in colon cancer.Screening of differentially expressed genes (DEGs) and pathways in the pathogenesis through constructing and analyzing high-throughput genomic data can contribute to the understanding on molecular mechanisms of colon cancer. These DEGs could act as new biomarkers for diagnosis and therapy of colon cancer [8]. In our previous studies, a pure population of colon cancer and normal colon cells was obtained and the global gene-expression differences were compared in the 2 cell types using combined cDNA microarray and antisense RNA-long serial analysis of gene expression (aRNA-LongSAGE) approaches. Several genes (IFITM3, FERMT1, MT1F, IMP3, PER 3, etc) that were differentially expressed in those 2 types of cells were identified as potential colon cancer related genes in further studies [9-13]. Eukaryotic translation initiation factor 3 subunit E (eIF3e) was overexpressed in colon cancer cells compared with normal colonic cells in our microarray data (signal:394.8 vs.124.9) and LongSAGE analysis (tag count:49 vs.14).Objective:The translation initiation of eukaryotic proteins is a complex cellular process, which requires a series of proteins to be involved, and these proteins are known as eukaryotic initiation factors(elF). Compared with the original nuclear factors, there are a varity kinds of eukaryotic initiation factors that are complicated also, of which 12 kinds have been identified. The translation initiation of eukaryotic cells was accomplished by the interaction between the eukaryotic initiation factors and the different eukaryotic initiation factors and the ribosome, mRNA and tRNA. Therefore, the eukaryotic translation initiation process dependent more on the interaction between protein and protein, protein and RNA, rather than between RNA and RNA, compared to prokaryotic. In addition to participating in the process of eukaryotic translation initation, many eukaryotic initiation factors also have other functions. Translation of most mRNAs is controlled at the ratelimiting step of initiation, leading to the assembly of an elongation-competent 80S ribosome from 40S and 60S subunits. First, the 43S ribosome pre-initiation complex must form:this consists of the 40S small ribosomal subunit, the initiating methionyl tRNA (Met-tRNAi) and a group of eukaryotic initiation factors (eIFs), including eIF2 (fIG. 1). Next, the 43S ribosome complex is recruited to the 5’end of the mRNA, marked by an inverted m7GpppN cap. Association of the 43S ribosome complex with the cap is mediated by the cap-binding (initiation) complex, eIF4F. Also associated with eIF4F and involved in initiation are several other initiation factors, including the multisubunit complex eIF3, and mRNA binding proteins such as the polyA tail binding protein (PABP; also known as PABPC1)The mammalian eIF3 complex is composed of 10-13 proteins that are involved in bridging the interaction between the 43 S pre-initiation complex and the eIF4Fbound mRNA18. Its pivotal position in protein synthesis and multiple functions has led to numerous and sometimes opposing roles for eIF3 in cancer, which can be clarified by examining the individual contributions of its subunits. Several of the subunits form a core complex that is thought to be essential for protein synthesis, although other subunits are probably involved in the regulation of the eIF3 complex or promote eIF3 interaction with specific mRNAs. EIF3e is a component of the eukaryotic translation initiation factor 3 (eIF-3) complexes, which is an essential factor for initiation of protein synthesis in mammalian cells. Translational control plays key roles in the complex mechanism of cancer development and progression. However, the clinical significance of eIF3e in colon cancer remains to be elucidated. In order to study the role of eIF3 played in colon cancer, we designed this experiment.Materials and methods:Tissue specimens were obtained from 173 colon cancer patients who underwent colectomy between January 2001 and December 2003 at Shanghai Jiaotong University Affiliated First People’s Hospital, both the cancerous tissues and the surrounding mormal tissues are collected, and none of these patients received adjuvant therapy prior to the surgery. All the formalin-fixed, paraffin-embedded tissues were applied for histological and immunohistochemical analysis. All specimens were analyzed and diagnosed by two pathologists who were blinded to clinical information. Tumor staging were carried out according to the American Joint Committee on Cancer (AJCC) classification system based on the tumor size (T), lymph node involvement (N), and distant metastasis (M). The disease-free survival (DFS) was defined as the interval from the surgery to clinically or radiologically proven recurrence or metastasis while the overall survival (OS) was defined as the interval from initial surgery to death. The follow-up was carried out according to the National Comprehensive Cancer Network Practice guidelines and the end date was June 29,2008. Theχ2 test or Fisher’s exact test was used to estimate the statistical significance of difference between eIF3e expression and clinicopathological features. Kaplan-Meier method was used for analyzing patients’cumulative survival rate. Furthermore, univariate and multivariate Cox proportional hazard model was applied to evaluate the hazard ratios for the variables. Differences between experimental groups were analyzed by t-test or one-way analysis of variance (ANOVA) followed by Bonferroni test. We used the statistical software SPSS 19.0 (Chicago, IL, USA) to carry out all statistical analyses. A P-value of less than 0.05 was regarded to be statistically significant.Results:Bioinformatics results:Six elF subunits were significantly overexpressed through bioinformatics analysis of 2 transcriptome databases Both of these genes were upregulated, including eIF3e (P< 0.001). Among these subunits, eIF3B, eIF3I, eIF4E and eIF5A had been validated to be associated with tumor progression. The role of eIF3e in colon cancer progression was urgent to be elucidated. Quantitative real-time polymerase chain reaction (RT-PCR) revealed that the eIF3e was over-expressed at mRNA level in tumor tissues. Thirty five of forty tumor tissues showed higher eIF3e mRNA expression compared with paired normal tissues. The ration of eIF3e mRNA expression was 3.04±2.65 and the difference of eIF3e mRNA expression between tumor tissues and paired normal tissues was significant (P< 0.01). TMA containing cancerous tissues paired with normal mucosa and lymph node metastasis was constructed and immunohistochemstry was performed to investigate the eIF3e protein expression in colon cancer. EIF3e was mainly observed brown staining in the cytoplasm of colon cancer cells, colon epithelial and mesenchymal Significant differences were found between normal mucosa and cancerous tissue (P< 0.001), normal mucosa and LNM metastasis (P< 0.001). Only 6 of 173 (3.5%) normal mucosa showed strong eIF3e expression with an obvious contrast to 42 of 173 (24.3%) in cancerous tissue and 31 of 54 (57.4%) in LNM tissue.Relationship of eIF3e expression with clinicopathological features in colon cancer: The relationship between eIF3e expression and clinicopathological features is displayed in. As shown, we found no significant differences between eIF3e expression and age, gender or tumor location (P> 0.05). On the contrary, eIF3e expression was significantly correlated with T classification (P< 0.001), lymph node involvement (P< 0.001), distant metastasis (P< 0.001), clinical stage (P< 0.001), dif-ferentiation (P< 0.001), and vessel invasion (P= 0.036). These data strongly indicated that eIF3e expression play an important role in the progression of colon cancer.Prognostic value of eIF3e expression in colon cancer patients:Kaplan-Meier curve analysis indicated that patients with high eIF3e expression were associated with poor prognosis. The estimated mean OS was 86.5 months,72.67 months and 37.1 months in patients with negative, weak and strong eIF3e expression, respectively. Similar results were observed while analyzing DFS in patients with different eIF3e expression (84.33 months,66.69 months and 29.14 months). To further evaluate the prognostic value of eIF3e expression in colon cancer patients, we performed univariate analysis for clinicopathological features and eIF3e expression level. In univariate an-alysis, lymph node in-volvement, distant me-tastasis, clinical stage, histopathological gra-de, vessel invasion and eIF3e expression were associated with both DFS and OS. Mul-tivariate analysis was carried out using the Cox proportional hazards model for significant variables in univariate analysis. N stage and M stage were excluded from the multivariate analysis for their collinearity with AJCC stage.High eIF3e expression remained significantly prognostic for decreased survival and increased disease recurrence. Strong eIF-3e expression was associated with app-roximately 10.3-fold risk of disease recurrence than negative eIF3e expression. Me-anwhile, patients with strong eIF3e suffer nearly 17-fold risk of decreased survival than those with negative eIF3e expression.Down-regulation of eIF3e in vitro inhibited colon cancer cell proliferation, clonality and promoted cell apoptosis.Conclusions:According to our digital gene expression profiling data by LCM-LongSAGE,6 eIF subunits (eIF2G, eIF3B, eIF3E, eIF3I, eIF4E and eIF5A) were significantly overexpressed in colon cancer cells. The contribution of eIF4E to cellular transformation, tumorigenesis and metastasis has been well documented over the past few years. EIF-4E was overexpressed in a variety of human tumors, and has been considered as a therapeutic target and as a marker for human cancer progression. Decreased expression of EIF3B gene resulted in the inhibition of human colon cancer cell proliferation. Overexpression of eIF3I drives colon oncogenesis by directly upregulating the synthesis of cyclooxygenase-2 (COX-2) protein and activating the β-catenin signaling pathway. The EIF5A mRNA expression level was a significant prognostic factor in CRC patients. The function and clinical significance of eIF2G in cancer has not been reported. We can infer that the abnormal expression of eIF subunits was an important molecular event in colon cancer progression.Regulation of the activity of eIF3 by its subunits resulted in hypo-and hyperactivation of the rate of the protein synthesis. A suitable level of translation initiation is necessary to regulate proliferation and cell cycle and contributes importantly to oncogenic transformation. Tumorigenesis in diverse tissue types is often associated with alterations in the expression of various eIF3 subunits. The expression and function of eIF3e, an up-regulated gene in our expression profiling database, is not yet characterized in colon carcinoma. In the present study, the elevated expression of elF3e at protein level in colon adenocarcinoma tissues was validated by immunohistochemistry staining. The overexpression of eIF3e protein was signifi-cantly associated with worse overall survival time and disease free survival time of colon cancer patients. Knockdown of eIF3e inhibits proliferation and promotes apoptosis in colon cancer cells. To our knowledge, this is the first study showed that eIF3e might be a colon cancer-related gene and its value as a potential novel prognostic marker of the disease.As a component of the eIF3 translation initiation factor, the pattern of protein synthesis changes mediated by eIF3e abnormal expression should be clarified in carcinogenesis. Recently, a microarray analysis revealed that mRNAs regulated either positively or negatively by eIF3e in human breast cancer cells were markedly enriched in genes associated with cell proliferation, invasion and apoptosis. In addition, RT-PCR analysis of RNA immunoprecipitated using an eIF3b-specific anti-body showed that apoptotic regulator BCLXL mRNA was decreased and the mitotic checkpoint component MAD2L1 mRNA enriched following eIF3e knockdown in MDA-MB-231 cells. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. These data suggest that eIF3e has an oncogenic role in breast cancer progression. The target genes regulated by eIF3e in colon cancer cells should be screened and validated in further study.The potential mechanisms of eIF3e in tumorigenesis were explored in some studies. EIF3e was identified as a tissue-specific modulator of MEK-ERK signaling through genetic and chemical-genetic approaches. The MEK protein levels reduced in the eIF3e-siRNA transfected cells. MEK, a dual specificity kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. Aberrant regulation of the MEK-ERK pathway can contribute to uncontrolled cell growth and lead to malignant transformation. The small-molecule targeting the MEK-ERK pathway was potential method for the treatment of cancer. A study showed that eIF3e suppression affects cell proliferation, cell cycle and apoptosis of various Glioblastomas (GBM) cells. These phenotypes are independent of global cell translation inhibition and are accompanied by decreased HIF expression when eIF3e is silenced. Further study should been made to clarify the precise role of eIF3e in colon cancer progression, and all the study may provide important diagnostic and therapeutic value of eIF3e in colon cancer patients in the future.