Journal: Theranostics

Article Title: Betaine inhibits the stem cell-like properties of hepatocellular carcinoma by activating autophagy via SAM/m 6 A/YTHDF1-mediated enhancement on ATG3 stability

doi: 10.7150/thno.102682

Figure Lengend Snippet: Betaine promotes ATG3 mRNA stability via SAM/m6A/YTHDF1-dependent manner. (A) After treatment with betaine, HCC cells were treated with 5 μg/mL of Act D for 0, 3, and 6 h, respectively. The expression levels of ATG3 mRNA at these different time points were detected using qPCR assay. (B) After pre-transfection with siBHMT, HCC cells were subjected to betaine or combined betaine and SAM treatments. Then, 5 μg/mL of Act D was added for further treatment for 0, 3, and 6 h, respectively. The expression levels of ATG3 mRNA at these different time points were detected using qPCR assay. (C) The correlation between ATG3 and YTHDF1 in HCC tissues was assessed using online GEPIA 2 database. (D) The interaction between ATG3 mRNA and YTHDF1 protein was analyzed using RIP-qPCR assay, and the RIP-derived protein and ATG3 mRNA in HCC cells were detected by WB and qPCR assays, respectively. (E) The interaction between ATG3 mRNA and YTHDF1 protein was confirmed by RNA pull-down assay, and the protein derived from the RNA pull-down assay in HCC cells was detected using WB assay. (F) Schematic graph of the YTHDF1 wild-type (YTHDF1-WT) and the mutant of YTHDF1 m 6 A-binding pocket (YTHDF1-MUT) construction. (G) HCC cells were pre-transfected with YTHDF1-WT and YTHDF1-MUT overexpressed plasmids, and the interaction between ATG3 mRNA and YTHDF1 was analyzed via RIP-qPCR assay. (H and I) HCC cells were transfected with YTHDF1-WT and YTHDF1-MUT overexpressed plasmids, and the expression levels of ATG3 mRNA as well as the levels ATG3 and YTHDF1 proteins were detected using qPCR and WB assays, respectively. (J and K) HCC cells were transfected with siYTHDF1, and the expression levels of ATG3 mRNA as well as the levels of ATG3 and YTHDF1 proteins were detected using qPCR and WB assays, respectively. (L-M) HCC cells were pre-transfected with siYTHDF1, and then were subjected to betaine treatment. The expression levels of ATG3 mRNA and protein were detected using qPCR and WB assays, respectively. (N) After pre-transfection with siYTHDF1, HCC cells were subjected to betaine treatment. Then, 5 μg/mL of Act D was added for further treatment for 0, 3, and 6 h, respectively. The expression levels of ATG3 mRNA at these different time points were detected using qPCR assay. **P < 0.01, ***P < 0.001.

Article Snippet: To validate that the binding of YTHDF1 protein and ATG3 mRNA was m 6 A dependent, the YTHDF1 specific m 6 A-recognition site mutant lentivirus was constructed by GenePharm (YTHDF1-MUT), and the wild type of YTHDF1 (YTHDF1-WT) was used as control ( ).

Techniques: Expressing, Transfection, Derivative Assay, Pull Down Assay, Mutagenesis, Binding Assay

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 1. Epi-Drug CRISPR dropout screens identify RUVBL1/2 as vulnerabilities of YTHDF1-expressing colorectal cancer cells. A, Composition of Epi-Drug sgRNA library and the workflow of CRISPR-Cas9 screens to identify YTHDF1-dependent vulnerabilities in colorectal cancer cells. B, Principal component analysis (PCA) of sgRNA abundances in each group at the end point of CRISPR-Cas9 screening. C, Left, top depleted genes in YTHDF1-overexpressing DLD1 cells vs. control vector (log2(fold change) < 0.5; log10(P value < 1). Middle, top enriched genes in shYTHDF1 cells vs. shControl (log2(fold change) > 0.5; log10(P value < 1). Right, overlapping of outlier genes identified the common candidates preferentially essential in a YTHDF1-dependent fashion. D and E, RUVBL1/2 mRNA expression in colorectal cancer cells compared with adjacent normal tissues in Hong Kong (D) and TCGA (E) colorectal cancer cohorts. In Hong Kong cohort, mRNA expression was normalized to β-actin. F, RUVBL1/2 and YTHDF1 proteins are overexpressed in colorectal cancer cells compared with paired adjacent normal tissues. G, Left, representative images of YTHDF1, RUVBL1, and RUVBL2 staining in colorectal cancer tissue microarrays (N ¼ 184). Right, Pearson correlation analysis of YTHDF1, RUVBL1, and RUVBL2 protein expression. H, Left, Kaplan–Meier curve analysis of RUVBL1 protein expression and patient survival in colorectal cancer in tissue microarray cohort (N ¼ 184). Right, multivariate Cox regression analysis. RUVBL1-low, IHC score 1; RUVBL1-high, IHC score 2 to 3. I, Left, Kaplan–Meier curve analysis of RUVBL2 protein expression and colorectal cancer patient survival. Right, multivariate Cox regression analysis. RUVBL2-low, IHC score 1 to 2; RUVBL2-high, IHC score 3. Paired t test (D and E; left), Student t-test (E; right), Pearson χ2 test (G), or log rank test (H and I).

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: CRISPR, Expressing, Control, Plasmid Preparation, Staining, Microarray

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 2. RUVBL1/2 knockout abolishes oncogenic function of YTHDF1 in vitro and in vivo. A–D, Effect of RUVBL1/2 knockout on vector- and YTHDF1-overexpressing DLD1 and HCT116 cell proliferation (N ¼ 10; A), colony formation (N ¼ 3, 7–14 days; B), apoptosis (N ¼ 3; C), and G1-S cell cycle transition (N ¼ 3; D). E, Western blot of cell cycle and apoptosis markers. F, Representative brightfield images of primary colorectal cancer tumor-derived organoids expressing vector or YTHDF1, with or without RUVBL1/2 knockout. G, Effect of RUVBL1/2 knockout on vector- and YTHDF1-overexpressing DLD1 and HCT116 xenografts in nude mice. RUVBL1/2 abrogated differential growth between vector- and YTHDF1-overexpresing xenografts (DLD1, N ¼ 5; HCT116, N ¼ 8). Two-way ANOVA (A) and one-way ANOVA (B–D and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Knock-Out, In Vitro, In Vivo, Plasmid Preparation, Western Blot, Derivative Assay, Expressing

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 3. YTHDF1 directly targets m6A-modified RUVBL1/2 mRNA methylation and promotes their protein expression in vitro and in vivo. A, UCSC snapshots of m6A-seq reads of RUVBL1/2 mRNA in DLD1 cells. The normalized read densities are shown for m6A (orange) and input (blue). B, Methylated RIP-qPCR analysis of m6A-modified RUVBL1/2 mRNA in DLD1 and HCT116 cells. C, RIP-qPCR with anti-YTHDF1 antibody showed binding of YTHDF1 to RUVBL1/2 mRNA, whereas mutant YTHDF1 (K395A, Y397A) had attenuated binding. D and E, Effect of YTHDF1 overexpression (D) or knockdown (E) on RUVBL1/2 mRNA and protein expression in DLD1 and HCT116 cells. F, Effect of YTHDF1 overexpression on RUVBL1/2 protein expression in primary colorectal cancer organoids PDO828 and PDO74. G, Expression of YTHDF1 and RUVBL1/2 in intestinal-specific Ythdf1 knockin mice (Ythdf1lslCdx2-CreERT2) as compared with wildtype mice. Student t test (B–D) and one-way ANOVA (E). ****, P< 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Modification, Methylation, Expressing, In Vitro, In Vivo, Binding Assay, Mutagenesis, Over Expression, Knockdown, Knock-In

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 4. YTHDF1 promotes translation efficiency of RUVBL1/2, which in turn interact with YTHDF1 and translational initiation factors. A, RNC-qPCR analysis of ribosome- associated RUVBL1/2 mRNA in vector- and YTHDF1-overexpressing DLD1 and HCT116 cells. B, Enrichment of RUVBL1/2 mRNA in < 40S, 40S, 60S, 80S, and polysomes from HCT116 cells with or without YTHDF1 overexpression. C and D, Colorectal cancer cells overexpressing wildtype YTHDF1 or mutant YTHDF1 were transfected with pmirGLO-RUVBL1 (C) or pmirGLO-RUVBL2 (D) containing respective 30UTR sequences, followed by luciferase assays. E, pmirGLO-RUVBL1/2-mutant reporters with mutated m6A sites (RRACH to TTTCT) in the 30UTR region demonstrated decreased luciferase activity. F, RUVBL1/2 coimmunoprecipitation and mass spectrometry for identification of common interacting proteins. G, Pathway enrichment analysis [gene ontology (GO), GSEA-KEGG] of interacting partners of RUVBL1/2. H, Coimmunoprecipitation by anti-YTHDF1 verified binding of YTHDF1 to RUVBL1/2. I, Coimmunoprecipitation using recombinant YTHDF1 and RUVBL1/2 confirmed direct protein–protein interplay between YTHDF1 and RUVBL1/2. J, Colocalization of RUVBL1/2 and YTHDF1 in HCT116 cells was determined by immu- nofluorescence staining. Student t test (A, B, and E) and one-way ANOVA (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Plasmid Preparation, Over Expression, Mutagenesis, Transfection, Luciferase, Activity Assay, Mass Spectrometry, Binding Assay, Recombinant, Staining

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 5. RUVBL1/2 knockout abrogated YTHDF1-induced translation initiation and oncogenic signaling. A, Left, polysome profiling of HCT116 cells with overexpression of YTHDF1 with or without knockout of RUVBL1/2. Right, Western blot of ribosomal fractions (<40S, 40S, 60S, 80S and polysomes). B, Stress granules (SG) were determined by immunofluorescence staining of TIA1-related protein (TIAR). C, HPG protein incorporation assay for the detection of nascent protein synthesis by immunofluorescence staining. D, Puromycin incorporation assay of protein synthesis. E, Ribo-seq of YTHDF1-overexpressing HCT116 cells with or without RUVBL1/2 knockout, following GSEA-KEGG pathway enrichment analysis. F, Effect of RUVBL1/2 knockout on the translation efficiency of MAP3K2, MAP3K7, MAPK8IP1, and ETS2 in HCT116 cells with YTHDF1 overexpression. G, Western blot of MAPK and PI3K-Akt signaling markers. One-way ANOVA (B and C). ***, P < 0.001; ****, P < 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Knock-Out, Over Expression, Western Blot, Immunofluorescence, Staining

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 6. Pharmacological RUVBL1/2 inhibitor inhibits the growth of YTHDF1-overexpressing colorectal cancer cells. A, Structure of a RUVBL1/2 complex inhibitor, CB6644. B, Forty-eight hours-IC50 values indicated that CB6644 preferentially inhibited the growth of DLD1 and HCT116 cells with YTHDF1 overexpression. C, CB6644 preferentially impaired colony formation capacity in YTHDF1-overexpressing DLD1 and HCT116 cells (7–14 days). D, CB6644 (0.5 µmol/L for DLD1; 0.1 µmol/L for HCT116, 24 hours) abrogated suppressive effect of YTDHF1 overexpression on apoptosis. Puromycin (0.5 µg/mL, 24 hours) was used as positive control. E, Treatment of DLD1 cells with CB6644 (0.5 µmol/L, 36 hours), followed by coimmunoprecipitation to analyze their interactions with YTHDF1. F, Interaction between YTHDF1 and EIF3K or EIF4A after treatment with CB6644 in DLD1 cells (0.5 µmol/L, 36 hours). G, Effect of CB6644 on protein translation in DLD1 cells, as assessed by puromycin incorporation assay (0.5 µmol/L, 6 hours). H, DLD1 cells expressing sgRUVBL1 or sgRUVBL2 were overexpressed with wildtype or ATPase-dead mutant RUVBL1 or RUVBL2, respectively. Coimmunoprecipitation was performed with anti-YTHDF1 to determine its interaction with RUVBL1/2, EIF3K, and EIF4A. I, Effect of ATPase-dead mutant RUVBL1 or RUVBL2 on protein translation in DLD1 cells compared with wildtype counterparts. One-way ANOVA (E and F). ****, P < 0.0001.

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: Over Expression, Positive Control, Expressing, Mutagenesis

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 7. In vivo efficacy of RUVBL1/2 inhibitors or vesicle-like nanoparticle-encapsulated siRUVBL1/2. A, DLD1 vector- or YTHDF1-overexpressing xenografts were treated with CB6644 (25 mg/kg, i.t.; arrows). B, HCT116 vector- or YTHDF1-overexpressing xenografts were treated with CB6644 (25 mg/kg, i.t.; arrows). C, Ki67 staining of DLD1 xenografts treated with CB6644. D, Structure of si-RUVBL1/2 encapsulated by VNPs. E, VNP-siRUVBL1/2 knockdown efficiency was confirmed in HCT116 cells in vitro. F, Effect of VNP-siRUVBL1/2 (2 mg/kg, i.t.; arrows) on DLD1 xenografts with or without YTHDF1 overexpression. G, Effect of VNP-siRUVBL1/2 (2 mg/kg, i.t.; arrows) on HCT116 xenografts with or without YTHDF1 overexpression. H, Ki67 staining of DLD1 xenografts treated with VNP-siRUVBL1/2. I, Schematic diagram showing the mechanism of RUVBL1/2 blockade in YTHDF1-expressing cells. RUVBL1/2 forms a complex with YTHDF1 and associated translation initiation factors, which is essential for YTHDF1-induced protein translation and oncogenic signaling. RUVBL1/2 themselves are targets of YTHDF1, forming a feedforward circuitry that boosts translation in colorectal cancer. RUVBL1/2 inhibition arrested translation by YTHDF1 and abrogated YTHDF1-induced oncogenic signaling and tumorigenesis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D and I, Created with BioRender.com.)

Article Snippet: YTHDF1 overexpression and knockdown in colorectal cancer cells Wildtype YTHDF1 or mutant YTHDF1 (K395A, Y397A) was cloned into pLentiCMV-Hygro. shRNAs were cloned into pLKO.1puro vector (RRID: Addgene_139470).

Techniques: In Vivo, Plasmid Preparation, Staining, Knockdown, In Vitro, Over Expression, Expressing, Inhibition