High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer

Abstract

The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser-capture microdissected cancer cells from responding and non-responding patients receiving XELOX/FOLFOX as first-line treatment for mCRC, and identified, among others, high expression of miR-625-3p, miR-181b and miR-27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first-line with XELOX, high expression of miR-625-3p was confirmed to be associated with poor response (OR = 6.25, 95%CI [1.8; 21.0]). Independent analyses showed that miR-625-3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR-625-3p solely is a response marker. Finally, we also found that these miRNAs were up-regulated in oxaliplatin resistant HCT116/oxPt (miR-625-3p, miR-181b and miR-27b) and LoVo/oxPt (miR-181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR-625-3p and response to first-line oxaliplatin based chemotherapy of mCRC.

Figure 1

Identification of miRNAs associated with response to treatment of mCRC. (A) Schematic overview of the strategy employed. (B) Volcano plot of 404 miRNA assays indicating a general up‐regulation in non‐responders (ΔCtNR – ΔCtR > 0, x‐axis) as compared to responders. Assays potentially associated with response to treatment (p < 0.05, Wilcoxon rank‐sum test, y‐axis, indicated with the horizontal line) are colored red. (C) miR‐185 normalized expression levels (ΔCt) of miR‐625‐3p, miR‐181b and miR‐27b in non‐responders (NR, n = 9) and responders (R, n = 17). Median ΔCt is indicated with a bar.

Figure 2

miR‐625‐3p, miR‐181b and miR‐27b are up‐regulated in oxaliplatin resistant colon cancer cells. (A) IC50 (left y‐axis) of oxaliplatin and corresponding relative resistance (right y‐axis) of HCT116 and HCT116/oxPt, and LoVo and LoVo/oxPt cell lines. (B) and (C) qRT‐PCR assays against individual miRNAs were done on isogenic parental and oxaliplatin resistant HCT116 (B) and LoVo (C) cell pairs. The expression levels in the resistant cells are plotted relative to the expression levels in the parental cells (set to 1). From each cell line two separate cell passages were obtained and from each passage qRT‐PCR was done twice and the average resistant/parental expression ratio from totally n = 4 qRT‐PCRs calculated, except for the unrelated miR‐93, miR‐192 and miR‐103, which were quantitated only once in each cell passage. S.E.M. is indicated, as well as significant different expression between parental and resistant isogenic cells (*, p < 0.05; t‐test).

Figure 3

miR‐625‐3p is up‐regulated in non‐responder patients in independent FFPE samples from XELOX treated mCRC patients. (A) Expression levels of miR‐625‐3p in validation Cohort B was significantly higher in non‐responder as compared to responder patients (t‐test). Mean expression and 95%CI is indicated with a diamond. (B) The proportions of patients that did not experience response to therapy (PD or SD) were higher in primary tumors with high miR‐625‐3p expression levels (n = 15) as compared to primary tumors with low miR‐625‐3p expression levels (n = 62) (Fischer's exact test). (C) Kaplan–Meier curves for OS and PFS according to high and low miR‐625‐3p expression levels (log‐rank test).

Figure 4

miR‐625‐3p is not a general marker for CRC. (A) The 24 miRNAs associated with response can separate tumor cells from normal mucosa epithelial cells. The miRNA name and corresponding p‐value for the difference in mean expression in normal vs. tumor using top 50‐normalized data is indicated (t‐test). Below are indicated normal (green), response (yellow) and no response (red) samples. (B) miR‐181b, but not miR‐625‐3p, is up‐regulated in cancer as compared to normal mucosa epithelium (t‐test). (C) Levels of miR‐625‐3p is similar in stage II and III tumors (right), as well as in tumors from recurrence and non‐recurrence Cohort C patients (left) (t‐test). (D) In non‐treated stage II Cohort D cancers no difference in miR‐625‐3p levels were found between recurrent and non‐recurrent patients (t‐test). In B, C and D mean expression and 95%CI is indicated with a diamond.