Fig 1: Targeting SMYD3 in patient-derived CRC-SCs to circumvent c-MYC mediated 5-FU chemoresistance. a Quantification of cell viability by CellTiter-Glo in patient-derived CRC-SCs pre-treated or not with EM127 (10 μM) for 48 h and then treated or not with 5-FU (10 μM) for 24 h. b Quantification of cell death by Trypan blue staining in patient-derived CRC-SCs treated as described in a. c Migratory ability of growth factor-starved patient-derived CRC-SCs placed in the inner chamber of transwell plates and treated or not with EM127 (10 μM) and/or 5-FU (10 μM) for 16 h. Migrating cells were fixed and counted under a fluorescence microscope. Scale bar: 100 μm. d Bar plot representation of the flow cytometry analysis of Ki67 expression in patient-derived CRC-SCs treated as described in a. e Bar plot representation of the flow cytometry analysis of annexin V staining in patient-derived CRC-SCs treated as described in a. The graph summarizes the percentage of apoptotic cells (early + late). f−h Analysis of tumors explanted from WT HCT-116 cell xenograft mice treated with EM127 (10 mg/kg daily) and/or 5-FU (25 mg/kg every 3 days) or the vehicle alone for 12 days. Tumor volume (data are expressed as means ± SD, each dot represents one mouse) (f), representative images (g), and hematoxylin and eosin (H&E) staining (h, scale bar: 100 μm). i–k Analysis of tumors explanted from patient-derived CRC-SC xenograft mice treated with EM127 (10 mg/kg daily) and/or 5-FU (25 mg/kg every 3 days) or the vehicle alone for 12 days. Tumor volume (data are expressed as means ± SD, each dot represents one mouse) (i), representative images (j), and hematoxylin and eosin (H&E) staining (k, scale bar: 200 μm). *p < 0.05 treated vs untreated; #p < 0.05 combined treatment vs corresponding single treatments. Where applicable, data are expressed as means ± SD of 3 independent experiments
Fig 2: In vivo studies to tailor a novel therapeutic approach based on SMYD3 inhibition. a Schematic illustration of the establishment of WT and SMYD3-KO HCT-116 cell xenograft mice. Analysis of tumors explanted from mice treated as depicted in a. b−d Tumor volume over time (b), hematoxylin and eosin (H&E) and Ki67 immunohistochemical staining (left panel) and bar plot summarizing the mean percentage of Ki67-positive cells (right panel) (c, scale bar: 100 μm), and immunoblot analysis of Cleaved PARP and Cleaved Caspase 3 levels (GAPDH was used as a loading control) (d). e Schematic illustration of the establishment of WT HCT-116 cell xenograft mice and EM127 treatment. As soon as the tumors reached a measurable size, mice were treated with daily intraperitoneal injections of EM127 (10 mg/kg) or the vehicle alone for 12 days and then sacrificed. f−h Analysis of tumors explanted from mice treated as depicted in e. Tumor volume over time (f), hematoxylin and eosin (H&E) and Ki67 immunohistochemical staining (left panel, scale bar: 100 μm) and bar plot summarizing the mean percentage of Ki67-positive cells (right panel) (g), and immunoblot analysis of Cleaved PARP and Cleaved Caspase 3 levels (GAPDH was used as a loading control) (h). i Schematic illustration of the establishment of AOM/DSS mice and EM127 treatment. After three cycles of DSS, mice were treated with daily intraperitoneal injections of EM127 (10 mg/kg) or the vehicle alone for 12 days and then sacrificed. j−l Analysis of tumors explanted from mice treated as depicted in i. Examination of explanted tissues showing tumor formation (j), average tumor number (k), and hematoxylin and eosin (H&E) and TUNEL staining (green) with nuclei counterstained with DAPI (blue) of colon sections (l, scale bar: 50 μm). AOM azoxymethane, DSS dextran sodium sulfate, IP intraperitoneal. b, c *p < 0.05 SMYD3-KO vs WT parental cells; f, g, k *p < 0.05 treated vs untreated. Where applicable, data are expressed as means ± SD of 3 independent experiments
Fig 3: Consensus molecular subtype (CMS) classification and gene set enrichment analysis (GSEA) hallmark pathways in patient-derived CRC-SCs. a Distribution of CMS groups in patient-derived CRC-SCs overexpressing SMYD3 (high-SMYD3) (n = 7) and not overexpressing SMYD3 (low-SMYD3) (n = 7). b GSEA results of the hallmark pathway in EM127-treated (5 μM for 24 h) vs untreated patient-derived CRC-SCs. The graphs represent the main hallmarks (y-axis) identified as significantly enriched in the high-SMYD3 and low-SMYD3 groups. The false discovery rate (FDR) Q value is also reported. c, d Dot plots of the top 20 ranked terms obtained from the Gene Ontology (GO) enrichment analysis (c) and the REACTOME analysis (d) of the MYC TARGETS V1 hallmark per the GSEA described in b. “Count” indicates the number of genes enriched in a GO term. “Gene ratio” indicates the percentage of enriched genes in the given GO term. e CancerMine (http://bionlp.bcgsc.ca/cancermine) classification of the c-MYC-downregulated and -upregulated targets identified as oncogenes (blue rectangles) or tumor suppressors (orange rectangle) in CRC
Fig 4: In vivo studies to address the potential of targeting SMYD3 in patient-derived CRC-SC metastatic mice models. a Schematic illustration of the establishment of patient-derived CRC-SC xenograft mice and EM127 treatment. Starting from day 1 after cell inoculation, mice were treated with daily intraperitoneal injections of EM127 (10 mg/kg) or the vehicle alone for seven days and sacrificed after six further weeks. b−e Analysis of tumors explanted from mice treated as depicted in a. Representative necroscopy showing numerous tumor masses (b), hematoxylin and eosin (H&E) staining with calcium salts visible in the red circle and TUNEL staining (c, scale bar: 50 μm), immunoblot analysis of Cleaved PARP and c-MYC K158/K163Me levels (GAPDH was used as a loading control) (d), and ddPCR analysis of stemness-related c-MYC target genes (e). f Schematic illustration of the establishment of patient-derived CRC-SC xenograft mice and EM127 treatment. Thirty days after cell inoculation, mice were treated with daily intraperitoneal injections of EM127 (10 mg/kg) or the vehicle alone for 12 days and then sacrificed. g–j Analysis of tumors explanted from mice treated as depicted in f. Representative necroscopy showing numerous tumor masses (g), hematoxylin and eosin (H&E) staining with calcium salts visible in the red circle and TUNEL staining (h, scale bar: 50 μm), immunoblot analysis of Cleaved PARP and c-MYC K158/K163Me levels (GAPDH was used as a loading control) (i), and ddPCR analysis of stemness-related c-MYC target genes (j). k, l Representative immunohistochemical analysis of c-MYC K158/K163Me levels in normal and cancer tissues from CRC patients (k, scale bar: 100 μm) and in normal tissue, primary tumor, and liver metastasis from metastatic CRC patients (l, scale bar: 100 μm). The TNM staging is indicated in parenthesis for each sample. *p < 0.05 EM127-treated vs untreated. Where applicable, data are expressed as means ± SD of 3 independent experiments
Fig 5: In vivo studies to address the potential of targeting SMYD3 in CRC preclinical metastatic mice models. a ddPCR analysis of EMT genes in SMYD3-KO vs WT HCT-116 tumorspheres. Data are expressed as means ± SD of 3 independent experiments. b Schematic illustration of the establishment of WT_luc and SMYD3-KO_luc HCT-116 cell xenograft mice. c, d Analysis of bioluminescence signal emission in whole animals treated as depicted in b. Bioluminescence imaging (BLI) average radiance measured over time (c) and images of bioluminescence emission by IVIS taken once a week for four weeks, with the intensity of photon emission being represented as a pseudo-color image (a representative scale bar is shown on the right) (d). e Representative necroscopy of mice treated as depicted in b showing numerous tumor masses (yellow circles). f Hematoxylin and eosin (H&E) and Ki67 immunohistochemical staining (left panel, scale bar: 100 μm) and bar plot summarizing the mean ± SD percentage of Ki67-positive cells (each dot represents one mouse, right panel) of tumors explanted from mice treated as depicted in b. *p < 0.05 SMYD3-KO vs WT parental cells
Supplier Page from Sino Biological, Inc. for Human SMYD3 transcript variant 2 Gene ORF cDNA clone in cloning vector