Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

Abstract

Colorectal cancer (CRC) has a high mortality rate and poor prognosis. Despite chemotherapeutic agents such as cisplatin, which has achieved a better prognosis and survival rate against cancer, drug resistance leads to significant challenges. Accumulating evidence suggests that YTHDF1, the N ⁶-methyladenosine (m6A) "reader," is an important regulator in tumor progresses. Herein, we report that YTHDF1 was significantly upregulated in human colon tumors and cell lines. Overexpression of YTHDF1 decreased the cisplatin sensitivity of colon cancer cells. From the established cisplatin-resistant CRC cell line (LoVo CDDP R), we detected that YTHDF1 was significantly upregulated in cisplatin-resistant CRC cells. Intriguingly, RNA sequencing (RNA-seq) results revealed that glutamine metabolism enzymes were clearly upregulated in LoVo CDDP R cells. Glutamine uptake, that is, glutaminase (GLS) activity, was upregulated in LoVo CDDP R cells. Furthermore, bioinformatics analysis indicated that the 3' UTR of GLS1 contained a putative binding motif of YTHDF1, and an interaction was further validated by a protein-RNA interaction assay (RNA immunoprecipitation [RIP]). Furthermore, we demonstrated that YTHDF1 promoted protein synthesis of GLS1. Inhibiting GLS1 effectively synergizes with cisplatin to induce colon cancer cell death. Finally, that YTHDF1 mediated cisplatin through the GLS1-glutamine metabolism axis was validated by an in vivo xenograft mouse model. In summary, our study reveals a new mechanism for YTHDF1-promoted cisplatin resistance, contributing to overcoming chemoresistant colon cancers.

Graphical abstract

Figure 1

Increased YTHDF1 expression is associated with CRC (A) Expression levels of YTHDF1 from 50 colon tumor tissues and their matched normal colon tissues were detected by qRT-PCR. (B) YTHDF1 protein expression levels were detected from eight CRC tumors and their matched normal colon tissues by western blot. β-Actin was a loading control. (C) Representative immunohistochemical staining of YTHDF1 protein expression levels from colon tumor tissues and their matched normal colon tissues. (D) mRNA expression levels of YTHDF1 in normal colon epithelial cells and five CRC cell lines were detected by qRT-PCR. (E and F) DLD-1 (E) and LoVo (F) cells were transfected with control or YTHDF1 overexpression plasmid for 48 h, followed by cisplatin treatments at the indicated concentrations. Cell viability was determined by MTT and clonogenic assays. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

Positive correlation between YTHDF1 and cisplatin resistance in colon cancer cells (A) LoVo CDDP R cells were established according to the description in Materials and methods. (B and C) LoVo parental and CDDP R cells were treated with cisplatin at the indicated concentrations for 48 h, and cell viability was measured by an MTT assay, clonogenic assay (B), and annexin V assay (C). (D and E) The protein (D) and mRNA (E) expression levels of EGFR were measured in DLD-1 parental and 5-fluorouracil (5-Fu)-resistant cells. (F) YTHDF1 was stably knocked down in LoVo CDDP R cells. Cell were treated with cisplatin at the indicated concentrations for 48 h, and cell viability was measured by an MTT assay. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Cisplatin-resistant CRC cells exhibit elevated glutamine metabolism (A) Heatmap of differentially expressed genes from LoVo parental and CDDP R cells identified by RNA-seq. (B) MetaboAnalyst pathway enrichment analysis of metabolites in LoVo parental and CDDP R cells. (C) mRNA expression levels of glutamine metabolism enzymes and regulators were detected by qRT-PCR in LoVo parental and CDDP R cells. (D) Western blot results show protein expression of GLS1 and GLS2 in LoVo parental and CDDP R cells. (E and F) Glutamine uptake (E) and GLS activity (F) assays were performed in LoVo parental and CDDP R cells. (G) LoVo parental and CDDP R cells were cultured with regular medium or glutamine depletion medium, a clonogenic assay and (H) annexin V assay were performed. (I) LoVo CDDP R cells cultured with regular medium or glutamine depletion medium were treated with cisplatin at 10 and 20 μM for 48 h. (J) Cell viability and cell death were examined by a clonogenic assay and (H) annexin V assay. Columns include mean of three independent experiments; data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

YTHDF1 directly binds with GLS1 to promote its protein translation (A and B) LoVo and DLD-1 cells were transfected with control, wild-type (WT) YTHDF1, or YTH domain mutant YTHDF1 for 48 h (A), and glutamine uptake and GLS activity (B) were detected. (C) Predicted YTHDF1 binding motif on the 3′ UTR of GLS1 mRNA. (D) RNA immunoprecipitation (RIP) was performed in LoVo and DLD-1 cells using anti-IgG control or anti-YTHDF1 antibody. (E and F) GLS1 abundance in the immunoprecipitated fraction was measured by agarose gel electrophoresis and (E) qRT-PCR. (F) An RNA pull-down assay was performed in LoVo and DLD-1 cells. The biotin-labeled 3′ UTR of WT or binding motif mutant GLS1 was incubated with proteins extracted from cells. The YTHDF1 protein, which was pulled down by the GLS1 binding motif, was detected by western blot. β-Actin was used as a negative control. (G) LoVo and DLD-1 cells were transfected with control shRNA or YTHDF1 shRNA for 48 h, and RIP experiments were performed using an anti-IgG control or anti-YTHDF1 antibody. GLS1 and β-actin mRNA abundance levels in the immunoprecipitated fraction were measured by agarose gel electrophoresis and (H) qRT-PCR. (I and J) Reporter constructs containing the WT or the binding motif mutant (Mut) GLS1 3′ UTR were co-transfected with control shRNA or GLS1 shRNA into LoVo and DLD-1 cells. Luciferase activities were measured using a Dual-Luciferase reporter assay kit. (K) LoVo and DLD-1 cells were treated with 10 μg/mL CHX for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. (L) LoVo and DLD-1 cells were treated with 50 nM MG-132 for 0 and 6 h, and the relative GLS1 protein expression was measured by western blot. β-Actin was used as an internal control. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Inhibiting GLS effectively synergizes with cisplatin to induce colon cancer cell death (A) DLD-1 cells were transfected with control shRNA or sh-GLS1 for 48 h, followed by treatments of cisplatin at the indicated concentrations. Cell viability was determined by MTT and (B) clonogenic assays. (C) LoVo cells were transfected with control shRNA or sh-GLS1 for 48 h, followed by treatments of cisplatin at the indicated concentrations. Cell viability was determined by MTT and (D) clonogenic assays. (E) DLD-1 cells were treated with BPTES at 0, 25, or 50 nM plus cisplatin at the indicated concentrations. Cell viability and cell death were examined by MTT and (F) annexin V assays. (G) The combination index (CI) was calculated using CompuSyn software based on the results of (E) and (F). (H) LoVo cells were treated with BPTES at 0, 25, or 50 nM plus cisplatin at the indicated concentrations. Cell viability and cell death were examined by MTT and (I) annexin V assays. (J) The CI was calculated using CompuSyn software based on the results of (H) and (I). Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

YTHDF1-mediated cisplatin resistance is through promoting glutamine metabolism (A) DLD-1 cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (B) The above cells were treated with cisplatin at 0, 2.5, 5, 10, 20, or 40 μM for 48 h. Cell viability and death were assessed by an MTT assay and (C) annexin V assay, respectively. (D) LoVo cells were transfected with control or YTHDF1 overexpression plasmid for 48 h. Cells were treated without or with BPTES for 24 h. The GLS activity was measured. (E) The above cells were treated with cisplatin at 0, 2, 4, 8, 16, or 32 μM for 48 h. Cell viability and death were assessed by an MTT assay and (F) annexin V assay, respectively. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

In vivo blocking YTHDF1-mediated glutamine metabolism sensitizes colon cancer cells to cisplatin (A) LoVo CDDP R cells were transfected with control shRNA or YTHDF1 shRNA. Cells were subcutaneously injected into nude mice for developing xenograft tumors. Mice without or with YTHDF1 silencing xenograft tumors were grouped and treated with control saline or cisplatin via intraperitoneal injection twice a week. Mice survival rates and (B) tumor growth were examined. (C) A total of 60 xenograft tumors from the above-treated mice of each experiment group (15 for each group). (D) Xenograft tumors from mice were dissected, and the protein expression levels of GLS1 were examined by western blot and quantified. Data are presented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001.