Abstract
Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥ 20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portions revealed that NTSR1 is frequently methylated in the adenomatous portion, while methylation levels are generally lower in the cancerous portions. These results suggest that NTSR1 methylation is associated with lateral and noninvasive growth of colorectal tumors, while low levels of methylation may contribute to the malignant potential through activation of NTSR1. Our data also indicate that NTSR1 methylation may be a prognostic biomarker in CRC.
Highlights
Colorectal cancer (CRC) is thought to arise through the accumulation of multiple genetic and epigenetic alterations, leading to the activation of oncogenes and loss of function of tumor suppressor genes [1]
We show that methylation-associated silencing of neurotensin receptor 1 (NTSR1) is inversely associated with the invasiveness of colorectal tumors, and that its methylation could be a predictive biomarker of a better prognosis in CRC patients
Unsupervised hierarchical clustering analysis of the methylated CpG island amplificationmicroarray (MCAM) data revealed a substantial number of differentially methylated genes (n = 575), the majority of which were methylated at higher levels in large noninvasive tumors (NTs) than in small invasive tumors (ITs) (Figure 1B, Supplementary Table S1)
Summary
Colorectal cancer (CRC) is thought to arise through the accumulation of multiple genetic and epigenetic alterations, leading to the activation of oncogenes and loss of function of tumor suppressor genes [1]. Spreading tumors (LSTs) are generally defined as lesions that extend laterally along the luminal wall and are greater than 10 mm in diameter with a low vertical axis [9]. These flat lesions are thought to be less invasive because they are more likely to be found at the adenoma stage or an early CRC stage [9,10,11]. One recent study reported that LST-G is characterized by an intermediate-methylation epigenotype and KRAS mutation, while LST-NG is associated with a low-methylation epigenotype and frequent CTNNB1 mutation [16]. An inverse association between APC methylation and submucosal invasion by LSTs has been reported [17]
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