Dichloroacetate Overcomes Oxaliplatin Chemoresistance in Colorectal Cancer through the miR-543/PTEN/Akt/mTOR Pathway

Abstract

Chemoresistance is responsible for most colorectal cancer (CRC) related deaths. In this study, we found that dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, can be used as a sensitizer for oxaliplatin (L-OHP) chemoresistant CRC cells. The aim of this study was to explore the ability of DCA to overcome L-OHP resistance in CRC cells and to identify the underlying molecular mechanisms. We found that DCA sensitizes chemoresistant CRC cells to L-OHP-induced cytotoxic effects by inhibiting clone formation capacity and promoting cell apoptosis. A microRNA (miRNA) array was used for screen, and miR-543 was identified and shown to be downregulated after DCA treatment. The expression of miR-543 was higher in chemoresistant CRC cells than in chemosensitive CRC cells. Overexpression of miR-543 increased chemoresistance in CRC cells. The validated target gene, PTEN, was negatively regulated by miR-543 both in vitro and in vivo, and PTEN was upregulated by DCA through miR-543. In addition, overexpression of miR-543 reversed the inhibition of colony formation after DCA treatment. Furthermore, the Akt/mTOR pathway is activated by miR-543 and is involved in the miR-543 induced chemoresistance. There was a significant inverse relationship between miR-543 expression and PTEN level in CRC patients, and high miR-543 expression was associated with worse prognosis. In conclusion, DCA restored chemosensitivity through miR-543/PTEN/Akt/mTOR pathway, and miR-543 may be a potential marker or therapeutic target for chemoresistance in CRC.

Keywords: chemoresistance; colorectal cancer; miR-543; miR-543 colorectal cancer; oxaliplatin.

Figure 1

DCA restores chemosensitivity in L-OHP resistant CRC cells. (A) HCT116, HCT-8, HCT-116/L, and HCT-8/L cells were treated with different concentrations of L-OHP, and cell survival was measured by CCK8. (B) The OD 450 determined by CCK8 was considered as the cell growth rate. (C-D) HCT116/L and HCT-8/L cells were treated with DCA or/and L-OHP for 24 hours. A colony formation assay was performed, and cell apoptosis was measured. The data shown are representative of three independent experiments. Mean ± SEM are presented, n = 3. *, P < .05; **, P < .01; ***, P < .001.

Figure 2

MiR-543 is downregulated after DCA treatment and is involved in chemoresistance. (A) Heatmap of differentially expressed microRNAs in HCT116 cells treated with 20 mM DCA for 24 hours. (B) Total RNA was prepared from HCT116 cells 24 hours after DCA treatment. Multiple microRNAs in microarray data sets were quantified by quantitative real-time PCR. (C) The basal level of miR-543 was determined by quantitative real-time PCR in L-HOP sensitive CRC cells and L-OHP resistant CRC cells. (D-F) HCT116 and HCT-8 cells transfected with miR-543 mimic or infected with overexpression virus were treated with or without L-OHP for 24 hours. The IC50 of L-OHP, the levels of c-PARP and Bax, and colony formation assays were analyzed. The data shown are representative of three independent experiments. Mean ± SEM are presented, n = 3. *, P < .05; **, P < .01; ***, P<.001; ns, no significance.

Figure 3

PTEN is modulated by DCA/miR-543 in CRC cells. (A) Examples of validated miR-543 targets and their biological functions. (B) The mRNA level of PTEN was determined by quantitative real-time PCR in HCT116 and HCT-8 cells treated with 20 mM and 15 mM DCA respectively for 24 hours. (C) Predicted structures of the potential binding site sequences of miR-543 with PTEN transcript by RNAhybrid. (D-E) HCT116 and HCT-8 cells were transiently transfected with miR-543 mimic or inhibitor. The mRNA and protein levels of PTEN were analysed. (F-G) HCT116 cells transfected with miR-543 mimic or NC were treated with or without DCA for 24 hours. The expression of PTEN and the colony formation assays were analysed. Representative results of three independent experiments are shown as the mean ± SEM, n = 3. *, P < .05; **, P < .01; ns, no significance.

Figure 4

MiR-543 promoted L-OHP chemoresistance in vivo. (A) Representative images of six tumor-bearing mice in each group before sacrifice and representative images of dissected tumors in each group (Scale bars: 1 cm). (B) Xenograft tumor volumes were calculated for 42 days after tumor inoculation. (C) Tumor weights of the two groups were measured. (D) The expression of miR-543 was determined by quantitative real-time PCR in xenograft tumors. (E-F) Immunostaining of xenograft tumor sections with Ki67 and PTEN. Positive staining is indicated by the brown colour (Scale bars: 100 µm) (left panel). The immunoreactive score was calculated (right panel). The mean ± SD is shown *, P < .05; **, P < .01; ***, P < .001.

Figure 5

Effects of miR-543 on the Akt/mTOR signaling pathways. (A) The protein levels of p-Akt, Akt, p-mTOR and mTOR in HCT116 and HCT-8 cells transfected with miR-543 mimics were detected by Western blot analysis. (B) Xenograft tumor sections were immunohistochemically stained for p-mTOR. Positive staining is indicated by the brown colour (Scale bars: 100 µm) (left panel). The immunoreactive score was calculated (right panel). The mean ± SD is shown, n = 6; **, P < .01. (C-D) Overexpression of miR-543 in HCT116 cells in the presence MK-2206 and/or rapamycin. p-mTOR (Ser 2448) expression, mTOR expression and the colony formation assays were analysed. (E) A diagram model depicting the mechanism in which DCA overcomes chemoresistance through the miR-543/PTEN/Akt/mTOR pathway.

Figure 6

The expression of miR-543 is negatively related to PTEN in CRC patients. (A) The relationship between the expression of miR-543 and PTEN was determined using linear regression analysis in CRC patients. (B) Human tumor and normal tissues were immunohistochemically stained with PTEN. Positive staining is indicated by a brown colour (Scale bars: 100 µm) (left panel). The immunoreactive score was calculated (right panel). (C) The OS of CRC patients was stratified by miR-543 or PTEN expression in TCGA datasets (P values were obtained using the log-rank test) (D) The mRNA level of PTEN in CRC and normal tissues in TCGA samples.