br We then tested the effect of CHFR knockdown on
We then tested the effect of CHFR knockdown on irinotecan sensitivity in CHFR-unmethylated cell lines (Figure 3, C and D). Both SNU-81 and CaCo-2 cell lines demonstrated loss of CHFR expression at 72 h after siRNA treatment (Figure 3C), and GI50 values were significantly lower in cell lines subjected to CHFR knockdown compared to their parental 2255-14-3 (Figure 3D).
In this study, we found that CHFR methylation was predictive of favorable treatment outcomes in terms of TTP in patients with metastatic CRC treated with irinotecan-based systemic chemother-apy. We further expanded our study in vitro and confirmed that CRC cell lines with CHFR methylation were more susceptible to irinotecan compared to those without methylation and that sensitivity to irinotecan could be modulated negatively by CHFR upregulation or positively by downregulating CHFR. In addition, irinotecan treatment outcomes differed according to CIMP status in patients with unmethylated CHFR. To the best of our knowledge, this is the first report on the clinical impact of CHFR solely focused on metastatic CRC and suggests CHFR methylation as a biomarker for irinotecan-based chemotherapy in patients with CRC.
CHFR was methylated in 15.7% of patients with metastatic CRC in our study, and this was lower than in previous studies reporting CHFR methylation in 31%-63% of patients, mostly with stage I-
III CRC [18,19,21,22,37]. When we analyzed the TCGA dataset, CHFR methylation levels did not differ by tumor stage in patients with CRC (P = .511 by ANOVA). Therefore, the lower prevalence of CHFR methylation in this study may be explained by differences in methylation assay or patient selection criteria. The correlation of CHFR with chemotherapy response or resistance has been mostly conducted for taxanes such as paclitaxel or docetaxel [23,24,27,38]. This is because CHFR is known to encode a checkpoint protein that delays entry into metaphase . Hence, the antitumor activity of microtubule inhibitors like taxanes could be boosted in cells with CHFR deficiency. In a previous study, the association of CHFR and chemotherapy response was specific to microtubule inhibitors but not to etoposide, a topoisomerase II inhibitor, or to cisplatin . In a study including 20 patients with CIMP-positive metastatic CRC who received nab-paclitaxel, however, treatment outcomes did not differ according to plasma CHFR methylation status, suggesting that CHFR methylation might not be predictive of taxane sensitivity, at least for patients with CRC. One potential explanation for the association between CHFR methylation and irinotecan response demonstrated in our study comes from the fact that CHFR was first described to function as an E3 ubiquitin ligase, which ubiquitinates and targets proteins for degradation by the S26 proteasome . One of the cellular mechanisms of irinotecan resistance is the repair of irinotecan-induced DNA damage . When the reversible Topo-I-irinotecan-DNA cleavable complex formed by irinotecan treatment collides with the advancing replication fork, Topo-I, the cellular target of irinotecan, is degraded through an ubiquitin/26S
proteasome-dependent system, and this facilitates the repair of single-strand breaks, thereby evading irinotecan-induced cellular damage. If the level of CHFR protein, a ubiquitin ligase, is decreased by DNA methylation, ubiquitination of Topo-I by 26S proteasome is impaired, and the subsequent upregulation of Topo-I could again render cancer cells more sensitive to irinotecan-induced cellular damage. This hypothesis is difficult to validate using clinical samples collected at a single time point and is also difficult to demonstrate using public data such as TCGA dataset. In fact, when we correlated CHFR methylation and TOP1 mRNA expression levels using the TCGA dataset, there was no apparent correlation between them (data not shown).
We demonstrated that CHFR methylation was closely associated with CIMP status in our clinical samples as well as in TCGA dataset. Although OS showed a trend favoring the CIMP-negative group (P =
.268 for OS after irinotecan treatment) in our cohort, the difference in TTP or OS based on CIMP status was not apparent in the overall population treated with irinotecan. We speculate that the different methodologies used in this study (MSP) and the previous one (MethyLight assay) or differences in patient selection criteria (irinotecan-based chemotherapy) may at least partially contribute to these results. However, in patients with unmethylated CHFR, TTP was poorer in the CIMP-positive group than in the CIMP-negative group, and multivariate analysis confirmed that the combination of CHFR/CIMP was a significant factor for both TTP and OS. Therefore, the significance of CHFR methylation in the treatment response to irinotecan may have weakened the prognostic impact of CIMP in our study cohort.