Advances in epigenetic biomarker research in colorectal cancer.

Colorectal cancer (CRC) causes approximately 600000 deaths annually and is the third leading cause of cancer mortality worldwide. Despite significant advancements in treatment options, CRC patient survival is still poor owing to a lack of effective tools for early diagnosis and a limited capacity for optimal therapeutic decision making. Since there exists a need to find new biomarkers to improve diagnosis of CRC, the research on epigenetic biomarkers for molecular diagnostics encourages the translation of this field from the bench to clinical practice. Epigenetic alterations are thought to hold great promise as tumor biomarkers. In this review, we will primarily focus on recent advances in the study of epigenetic biomarkers for colorectal cancer and discuss epigenetic biomarkers, including DNA methylation, microRNA expression and histone modification, in cancer tissue, stool, plasma, serum, cell lines and xenografts. These studies have improved the chances that epigenetic biomarkers will find a place in the clinical practices of screening, early diagnosis, prognosis, therapy choice and recurrence surveillance for CRC patients. However, these studies have typically been small in size, and evaluation at a larger scale of well-controlled randomized clinical trials is the next step that is necessary to increase the quality of epigenetic biomarkers and ensure their widespread clinical use.

A novel variant of the RON receptor tyrosine kinase derived from colorectal carcinoma cells which lacks tyrosine phosphorylation but induces cell migration.

Generation of splice variants in the RON receptor tyrosine kinase facilitates the invasive phenotype of colorectal cancers. Here, we report a new splice variant of RON in the human colorectal cancer cell line HCT116. This variant is encoded by a transcript differing from the full-length RON mRNA by an in-frame deletion of 106 amino acids in the extracellular domain of RON ß-chain. The deleted transcript originates by an alternative deletion of exon 2 and exon 3. The molecular weight of this variant is 160 kDa. Thus, we named this variant RONΔ160(E2E3). This variant is a single-chain protein and expressed in the intracellular compartment. We found that RONΔ160(E2E3) had no tyrosine phosphorylation ability, but it has constitutively activated Akt activity in transfected HEK293 epithelial cells. The expression of this variant in HEK293 cells resulted in an increased migratory activity in vitro mediated through the PI-3K/Akt pathway. Our data describes a new splice variant of RON and suggests a novel role for the RON receptor in the progression of metastasis in colorectal cancer.

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes.

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.

A novel HURRAH protocol reveals high numbers of monomorphic MHC class II loci and two asymmetric multi-locus haplotypes in the Père David's deer.

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.