Comprehensive molecular exploration identified promoter DNA methylation of the CRBP1 gene as a determinant of radiation sensitivity in rectal cancer

Abstract

Background: Neoadjuvant chemoradiotherapy (NCRT) for advanced rectal cancer (RC) is a well-evidenced therapy; however, some RC patients have no therapeutic response. Patient selection for NCRT so that non-responsive patients are excluded has been subjective. To date, no molecular markers indicating radiation sensitivity have been reported.

Methods: We irradiated six colorectal cancer (CRC) cell lines and identified HCT116 cells as radiation-sensitive and HCT15 and DLD-1 cells as radiation resistant. Using a microarray, we selected candidate radiation sensitivity marker genes by choosing genes whose expression was consistent with a radiation-resistant or sensitive cell phenotype.

Results: Among candidate genes, cellular retinol binding protein 1 (CRBP1) was of particular interest because it was not only induced in HCT116 cells by tentative 10 Gy radiation treatments, but also its expression was increased in HCT116-derived radiation-resistant cells vs parental cells. Forced expression of CRBP1 decreased the viability of both HCT15 and DLD-1 cells in response to radiation therapy. We also confirmed that CRBP1 was epigenetically silenced by hypermethylation of its promoter DNA, and that the quantitative methylation value of CRBP1 significantly correlated with histological response in RC patients with NCRT (P=0.031).

Conclusions: Our study identified CRBP1 as a radiation-sensitive predictor in RC.

Figure 1

Radiation sensitivity assay and establishment of radiation-resistant cells.(A) The protocol for selection of radiation-sensitive and resistant cells. (B) The ratio of the cell number in each cell line after to before radiation. (C) The protocol for establishment of radiation-resistant HCT116 cells. (D) Cell count after 5 Gy radiation for confirmation of the acquisition of radiation resistance following exposure of HCT116 cells to continuous RT to a total of 60 Gy.

Figure 2

Schematic outline of the definition of radiation sensitivity and resistance-related genes in microarray analysis.

Figure 3

Selection of candidate genes related to radiation sensitivity among the heat map genes.(A) Heat map of Affymetrix GeneChip gene expression microarray in radiation-resistant and -sensitive CRC cells. Red coloured bars indicate upregulated genes and green coloured bars indicate downregulated genes. (B) Relative results of RT–PCR analysis, which was performed to confirm microarray analysis results. (C) The selection criteria for sensitivity-related and resistance-related genes. (D) The results of RT–PCR for the selection of sensitivity-related genes. P, Parental cell; 10 Gy, after 10 Gy radiation; I, I-HCT116; M, M-HCT116 (Figure 1C) (E) The results of quantitative RT–PCR according to CRBP1 expression.

Figure 4

Changes of radiation sensitivity by the forced expression of CRBP1 gene.(A) The result of quantitative RT–PCR analysis of CRBP1 expression in CRC cell lines. P, Parental; R, after 10 Gy radiation. (B) RT–PCR and Western blotting to confirm CRBP1 expression in CRBP1-transfected HCT15 and DLD-1 cells. (C) Cell proliferation assay after CRBP1 transfection. (D) Cell proliferation assay after radiation of transfected CRC cell lines. (E) Cell counts after radiation of transfected CRC cell lines. (F) Scheme of CRBP1 function.

Figure 5

Analysis of promoter hypermethylation of CRBP1 gene in CRC cell lines and clinical samples.(A) Schematic diagram of CpG islands within the CRBP1 promoter region. TSS; translation start site. (B) Representative results of bisulfite sequencing of HCT15, DLD-1, and HCT116. The boxed arrow indicates methylated cytosine. (C) Methylation status of 26 individual CpG sites (columns) of 10 cloned PCR products (rows) within the CRBP1 promoter region by bisulfite sequencing. Open and filled circles, unmethylated and methylated CpG sites, respectively. Numbers in parenthesis denote TaqMeth V. (D) The result of RT–PCR analysis of CRBP1 after demethylation treatments in HCT15 and DLD1 cells. TSA, trichostatin A; Aza, 5-Aza-dC. (E) Correlation between expression and methylation status in CRC cell lines. (F) Distribution of the CRBP1 TaqMeth V in tumour tissue and corresponding normal mucosa. (G) Correlation between therapeutic response of 33 RC tissues and the TaqMeth V.