EphB3/ephrin-B分子訊息在人類大腸直腸癌之臨床和細胞分子生物學研究

Abstract

Colorectal cancer is one of the leading causes of cancer death in developed countries. Recently, targeted therapy has raised hopes for treating unresectable cancers, including colorectal cancer, more effectively with fewer side effects. Understanding the details of signaling transduction is prerequisite for development and clinical application of targeted therapy. The first part of this study aimed to find individual up-regulated genes responsible for clinicopathological variations in human colorectal cancer. The second part of this study aimed to disclose EphB3 gene function. We provided evidence demonstrating that EphB3/ephrin-B signaling was able to promote apico-basal polarization and epithelialization (promote mesenchymal- to-epithelial transition) – a new role for EphB3 in the biology of the gut and cancer, and activation of such signaling has great potential for targeted therapy. PART I. Clinical Correlates of Up-regulated Genes in Colorectal Cancer (A Forward or Classical Genetics Study) We hypothesize that changes in the transcription of up-regulated genes are biologically meaningful and may be linked to variations in tumor behavior and clinical features. Genes up-regulated concurrently in four microarray experiments were taken as candidate genes. Twenty candidate genes were verified using real-time RT-PCR in these 4 and another 27 pairs of samples. The presence or absence of up-regulation of these genes was correlated with ten clinicopathological variables from 31 patients. The mRNA transcript levels of these 20 candidate genes in the 31 paired samples were also correlated with each other to disclose any expression relationship. 40% (8/20) of candidate genes were verified by real-time RT-PCR to have a tumor/normal expression ratio > 2. Up-regulation of THY1 and PHLAD1 was associated with the presence of anemia in colon cancer patients (p=0.036 and 0.009). Up-regulation of HNRPA1 was more significant in cancer growing in the right sided colon than the left side (p=0.027). Up-regulated GPX2 was related to a higher degree of tumor differentiation (p=0.019). c-MYC was significantly over-expressed in specimens from male rather than female colon cancer patients (p=0.012). GRO1 was significantly up-regulated in patients younger than 65 years old (p=0.010) and was found to be frequently over-expressed when cancers were less invasive. In addition, we found normalized transcript levels of HNPRA1 were tightly associated with that of c-MYC (r=0.948). We conclude that validation of microarray data using another independent laboratory approach is mandatory and statistical correlation between gene expression status and patient’s clinical features may reveal individual genes relevant to tumor behavior and clinicopathological variations in human colorectal cancer. PART II. EphB3 Promotes MET and Suppresses Tumor Growth (A Reverse Genetics Study) Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in ApcMin/+ mice and EphB-mediated compartment- alization was demonstrated to be a mechanism suppressing colorectal cancer progress- ion; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4/ephrin-A and EphB4/ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3/ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes’ stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar, and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression reorganized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical-actin- cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased Transwell migration, increased apoptosis and Ca2+- dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear β-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL–Rac1, a known epithelial-to-mesenchymal transition (EMT) signaling pathway. Additionally, crosstalk from Wnt signaling potentiated the restora- tion of epithelial cell-polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3/ephrinB interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3- mediated tumor suppression.