br In the multivariate analyses
In the multivariate analyses for TTP and OS of irinotecan treatment, CHFR methylation was confirmed to be significantly associated with TTP after adjustment for potential confounding factors [adjusted HR, 2.88 (95% CI, 1.50-5.52), P = .001; Table 2], whereas methylation of WRN and SULF2, and the CIMP status were
not. Methylation of CHFR, WRN, SULF2, and the CIMP status were not significant factors in the multivariate analysis for OS.
As CHFR methylation was associated with CIMP-positive status, the association of CHFR/CIMP status was also analyzed for TTP and OS. TTP and OS were significantly different according to the CHFR/
Figure 2. (A) CHFR promoter methylation in 12 colorectal cancer cell lines as determined by methylation-specific PCR assays (upper panel). CHFR protein 2255-14-3 as determined by Western blotting (lower panel). (B) In vitro sensitivity of 12 colorectal cancer cell lines to increasing concentrations of irinotecan as determined by MTS cell proliferation assays. Cell growth curves (green, CHFR-methylated; red, unmethylated) are based on three independent experiments performed in triplicate (left panel). Nonlinear fit curves for each cell line are shown in black. GI50 values for each cell line are shown as bars representing the mean ± SEM (right panel). (C) Overexpression of CHFR in CHFR-methylated cell lines (HCT-116 and SNU-C5) was confirmed by Western blotting. (D) Irinotecan sensitivity after CHFR overexpression in HCT-116 and SNU-C5. Cell growth curves and GI50 values were determined at 48 and 72 hours after CHFR overexpression following treatment with increasing concentrations of irinotecan. Values are based on three independent experiments performed in triplicate.
152 CHFR Promoter Methylation in Metastatic Colorectal Cancer Cha et al. Neoplasia Vol. 21, No. 1, 2019
CHFR Methylation and In Vitro Sensitivity to Irinotecan
Irinotecan sensitivity in vitro based on CHFR methylation status was evaluated in 12 CRC cell lines. When CHFR promoter methylation status was assessed with MSP, six cell lines (RKO, HT-29, HCT-116, DLD-1, SNU-407, and SNU-C5) showed CHFR methylation, whereas five cell lines (CaCo-2, SW-620, SW-480, SNU-81, SNU-C4) had an unmethylated promoter (Figure 2A).
CHFR was partially methylated in LoVo cells. Western blot analysis revealed that protein expression levels of CHFR were inversely correlated with CHFR methylation status (Figure 2A). We confirmed the inverse correlation between CHFR methylation and mRNA expression in clinical samples as well using the TCGA dataset (Pearson correlation coefficient = −0.738, P b 0.001; Supplementary Material 5).
Cell proliferation assay demonstrated that CRC cell lines with CHFR methylation were more sensitive to irinotecan treatment compared to those with unmethylated CHFR (Figure 2B). Notably, all four cell lines with GI50 values b100 μM for irinotecan had methylated CHFR (RKO, SNU-C5, SNU-407, and HCT-116). We also compared the IC50 of 12 CRC cell lines according to CHFR methylation status using CCLE data (Supplementary material 6). Although the mean IC50 was lower in CHFR-methylated cells (n = 6) than in unmethylated cells (n = 6), the difference between groups was not significant (P = .441).
We then investigated the effect of CHFR overexpression on sensitivity to irinotecan in CHFR-methylated HCT-116 and SNU-C5 cells. After CHFR overexpression (Figure 2C), sensitivity to irinotecan measured at 48 and 72 hours was significantly diminished
Figure 3. (A) Western blotting for CHFR in CHFR-methylated cell lines (RKO and HCT-116) at 24 and 48 hours after 5-Aza-CdR treatment at 5 μM. (B) Irinotecan sensitivity in RKO and HCT-116 cells after 5-Aza-CdR treatment. Cell growth curves and GI50 values were plotted at 72 hours after 5-Aza-CdR treatment following treatment with increasing concentrations of irinotecan. (C) Western blotting for CHFR in CHFR-unmethylated cell lines (SNU-81 and CaCo-2) after CHFR knockdown with siRNA at 24, 48, and 72 hours. (D) Irinotecan sensitivity in SNU-81 and CaCo-2 cells after CHFR knockdown. Cell growth curves and GI50 values were plotted at 72 hours after CHFR knockdown following treatment with increasing concentrations of irinotecan. All values are based on three independent experiments performed in triplicate.
Neoplasia Vol. 21, No. 1, 2019 CHFR Promoter Methylation in Metastatic Colorectal Cancer Cha et al. 153
in both cell lines compared to their parental cells (Figure 2D). We also compared the viability of CHFR-methylated cell lines (RKO and HCT-116) with increasing concentrations of irinotecan along with or without 5-Aza-CdR (5 μM), a DNA-demethylating agent (Figure 3, A and B). Both RKO and HCT-116 cells showed CHFR upregulation after 5-Aza-CdR treatment (Figure 3A), and combination with 5-Aza-CdR decreased the growth inhibitory potential of irinotecan in all three cell lines (Figure 3B).