Genes with Aberrant Expression in Murine Preneoplastic Intestine Show Epigenetic and Expression Changes in Normal Mucosa of Colon Cancer Patients

Abstract

An understanding of early genetic/epigenetic changes in colorectal cancer would aid in diagnosis and prognosis. To identify these changes in human preneoplastic tissue, we first studied our mouse model in which Mthfr þ/À BALB/c mice fed folate-deficient diets develop intestinal tumors in contrast to MthfrBALB/c mice fed control diets. Transcriptome profiling was performed in normal intestine from mice with low or high tumor susceptibility. We identified 12 upregulated and 51 downregulated genes in tumor-prone mice. Affected pathways included retinoid acid synthesis, lipid and glucose metabolism, apoptosis and inflammation. We compared murine candidates from this microarray analysis, and murine candidates from an earlier strain-based comparison, with a set of human genes that we had identified in previous methylome profiling of normal human colonic mucosa, from colorectal cancer patients and controls. From the extensive list of human methylome candidates, our approach uncovered five orthologous genes that had shown changes in murine expression profiles (PDK4, SPRR1A, SPRR2A, NR1H4, and PYCARD). The human orthologs were assayed by bisulfite-pyrosequencing for methylation at 14 CpGs. All CpGs exhibited significant methylation differences in normal mucosa between colorectal cancer patients and controls; expression differences for these genes were also observed. PYCARD and NR1H4 methylation differences showed promise as markers for presence of polyps in controls. We conclude that common pathways are disturbed in preneoplastic intestine in our animal model and morphologically normal mucosa of patients with colorectal cancer, and present an initial version of a DNA methylation-based signature for human preneoplastic colon. Cancer Prev Res; 6(11);

Validation of Methylation Biomarkers that Distinguish Normal Colon Mucosa of Cancer Patients from Normal Colon Mucosa of Patients without Cancer

“…However, we used right side biopsies from the controls because we were attempting to validate candidate genes identified from right side biopsies, also reasoning that biomarkers that could distinguish cancer, independent of site within the colon would be more valuable, clinically. Although site-specific DNA methylation has been reported by us and others (14, 25, 27) to vary as a function of biopsy site within the colon for a number of genes, biopsy site does not appear to be a major factor at most CpG sites. Genome-wide comparison of methylation in right-side biopsies (from the ascending colon) and biopsies from the left side or rectum has identified both hyper- and hypo-methylation differences between the two anatomical sites (27).…”

“…We have described two methylation biomarkers that show some promise in distinguishing individuals without cancer or polyps from cancer-free individuals who have polyps (14). In this study, we attempted to define a support vector machine to classify which individuals in our group of controls carried polyps but its performance was mediocre: 12 of the controls had polyps (two with hyperplastic polyps, eight with tubular adenomas and two with polyps that were not described histopathologically) and 12 did not (Supplementary Table 1).…”

“…Because epigenetic changes play a strong role in colorectal cancer (5-10, e.g., also reviewed 11, 12), we (13,14), and others (15-17), have suggested that it may be possible to use quantitative objective measures of epigenetic change in normal tissues to detect colorectal cancer or precancerous lesions. In fact, we identified significant differences in methylation level at CpGs in 114-874 genes between the normal colon mucosa of 30 cancer patients and 18 controls by array-based methylation profiling (13).…”

Diet, Nutrition, and Cancer Epigenetics

“…Many additional studies in animal models have shown correlations between maternal diet and DNA methylation levels or histone modifications in offspring (1, 24,66,93). Correlations between epigenetic modifications and individual diet (as opposed to maternal diet during gestation) have also been provided by animal models (23,39,41,49,62,84,107,109), in some cases leading to mechanistic interpretations amenable to dietary intervention (28 …”

Emerging Technologies for the Clinical Microbiology Laboratory

“…Future clinical applications of pyrosequencing include transcriptome sequencing, which aims to efficiently create RNA profiles and examine the effects of mRNA transcript expression. The majority of research using NGS for transcriptome analysis has involved the basic sciences; however, recent studies have utilized this method for comparison of mRNA expression in normal and malignant cell populations and for discovery of latent or cryptic viruses whose presence and expression may be associated with malignancies (174)(175)(176).…”