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. 2015 Oct 6;6(30):29975-90.
doi: 10.18632/oncotarget.5034.

Epigenetic silencing of NTSR1 is associated with lateral and noninvasive growth of colorectal tumors

Seiko Kamimae  1 , Eiichiro Yamamoto  1   2 , Masahiro Kai  1 , Takeshi Niinuma  1   2 , Hiro-o Yamano  3 , Masanori Nojima  4 , Kennjiro Yoshikawa  3 , Tomoaki Kimura  3 , Ryo Takagi  3 , Eiji Harada  3 , Taku Harada  1 , Reo Maruyama  1   2 , Yasushi Sasaki  5 , Takashi Tokino  5 , Yasuhisa Shinomura  2 , Tamotsu Sugai  6 , Kohzoh Imai  7 , Hiromu Suzuki  1
Affiliations

Epigenetic silencing of NTSR1 is associated with lateral and noninvasive growth of colorectal tumors

Seiko Kamimae et al. Oncotarget. .

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.

Keywords: DNA methylation; LST; biomarker; colorectal tumor; invasion.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors disclose no conflict of interests.

Figures

Figure 1
Figure 1. Identification of NTSR1 methylation in large noninvasive tumors
A. Representative examples of a small invasive tumor (IT) and a large noninvasive tumors (NT). Endoscopic views are shown on the top and histological views are blow. B. Summary of MCAM results from small ITs, large NTs and normal colon. Genes differentially methylated between small ITs and large NTs were selected, after which unsupervised hierarchical clustering was carried out. Each row represents a single probe, and each column represents an average of each category. C. Diagram of the NTSR1 CpG island. The transcription start site and exon 1 are shown on the top, and the regions analyzed using bisulfite pyrosequencing, bisulfite sequencing and ChIP-PCR are shown below. D. Summarized results of the bisulfite pyrosequencing analysis of NTSR1 in specimens from the indicated colorectal tumor types and adjacent normal colonic tissue. E. Frequencies of NTSR1 copy number gain in the indicated types of colorectal tumors. The number of samples in each portion is also shown.
Figure 2
Figure 2. Analysis of NTSR1 methylation in colorectal tumors
A. Kaplan-Meier curves showing the effect of NTSR1 methylation (high, ≥ 29%; low, < 29%) on overall survival of patients with invasive colorectal tumors (n = 91). B. Summarized results of bisulfite pyrosequencing of NTSR1 in the indicated morphological types of noninvasive tumors.
Figure 3
Figure 3. Analysis of NTSR1 methylation and expression in CRC cell lines
A. Bisulfite pyrosequencing (top) and quantitative RT-PCR (bottom) analyses of NTSR1 in the indicated CRC cell lines and normal colonic tissue. B. Bisulfite sequencing analysis of the NTSR1 CpG island in the indicated CRC cell lines. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The arrow indicates the region analyzed with bisulfite pyrosequencing. C. RT-PCR results (left) and quantitative RT-PCR (right) analysis of NTSR1 in the indicated CRC cell lines, with or without 5-aza-dC treatment. Cells were treated with 2 μM 5-aza-dC for 72 h. D. ChIP-PCR analyses of NTSR1 in CRC cells. Levels of H3K4me3 and H3K27me3 in two regions around the NTSR1 transcription start site (see Figure 1C) are shown.
Figure 4
Figure 4. Functional analysis of NTSR1 in CRC cells
A. RT-PCR analysis of NTS in CRC cell lines and normal colonic tissue. B. Western blot analysis of NTSR1 in the indicated CRC cells transfected with a NTSR1 expression vector or a control vector (Vector). C. Colony formation assays using the indicated CRC cell lines transfected with a NTSR1 expression vector or a control vector. Representative results are on the left, and relative colony formation efficiencies are on the right. Shown are means of 3 replications; error bars represent SDs. D. Matrigel invasion assays using the indicated CRC cell lines transfected with a NTSR1 expression vector or a control vector. Invading cells are indicated by arrowheads. Shown on the right are the means of 5 random microscopic fields per membrane; error bars represent SDs. E. Results of quantitative RT-PCR analysis of NTSR1 expression (left), cell viability assays (middle) and Matrigel invasion assays (right) using HCT116 cells transfected with siRNA targeting NTSR1 (siNTSR1) or a control siRNA (siCONT). Values were normalized to cells transfected with the control siRNA. Cell viability assay results are means of 8 replications. Matrigel invasion assay results are means of 5 random microscopic fields per membrane. Error bars represent SDs. NS, not significant.
Figure 5
Figure 5. Alteration of NTSR1 methylation during the progression of colorectal tumorigenesis
A. Endoscopic view of a colorectal tumor consisting of an adenomatous portion (blue dot) and an invasive cancerous portion (red dot). B. Levels of NTSR1 methylation in biopsy specimens from the indicated portions analyzed by bisulfite pyrosequencing. C. Bisulfite sequencing analysis of NTSR1 in the indicated portions. The arrow indicates the region analyzed with bisulfite pyrosequencing. D. Array CGH results of the NTSR1 locus in the indicated portions. Each dot represents a single probe and genomic gains are indicated by dots on the right; losses are on the left. E. Immunohistochemical analysis of NTSR1. Magnified views of the cancerous and adenomatous portions (indicated by red and blue boxes, respectively) are shown below. F. Summarized results of bisulfite pyrosequencing of NTSR1 in colorectal tumors containing adenomatous and cancerous portions (n = 22). Methylation levels in adjacent normal colonic tissue from the same patients are also shown.
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