EIF5B/mitogen-activated protein kinase 1 axis drives tumor progression and confers dual resistance to chemotherapy and immunotherapy in colonic adenocarcinoma

Figure 1 Eukaryotic translation initiation factor 5B was upregulated in cancerous tissue and predicted a poor prognosis for patients with colonic adenocarcinoma. A-F: Analyses based on databases demonstrated significantly higher eukaryotic translation initiation factor 5B (EIF5B) mRNA expression in colonic adenocarcinoma (COAD) cancer tissues than in paracancerous tissues; G: Representative immunohistochemical staining of paired normal and tumor specimens from patients with COAD. EIF5B was undetectable in normal colonic tissue, whereas COAD tissues exhibited high EIF5B expression; H: Kaplan-Meier method was employed to assess the correlation of EIF5B expression with the prognosis of patients with COAD. All photomicrographs were captured at 200 × magnification. Independent-samples t-test, paired-samples t-test, and non-parametric tests were used for two-group comparisons. ^aP < 0.05. ^bP < 0.01. ^dP < 0.0001. TCGA: The Cancer Genome Atlas; COAD: Colonic adenocarcinoma.

Figure 2 Eukaryotic translation initiation factor 5B enhanced colonic adenocarcinoma cell proliferation and drove tumor growth. A: Correlation of individual genes with eukaryotic translation initiation factor 5B (EIF5B) expression was assessed using three independent datasets; B: Gene Ontology-Biological Process enrichment analysis of EIF5B was conducted using three independent datasets; C: mRNA levels of EIF5B in the colonic adenocarcinoma (COAD) cell lines; D-G: Assessment of EIF5B expression at mRNA and protein levels across transfected COAD cell lines; H and I: Proliferation of EIF5B-overexpressing COAD cells were investigated; J and K: Proliferation of EIF5B-knockdown COAD cells were investigated; L: Tumor growth curves (n = 6 per group); M: Representative tumor images in nude mice (n = 6 per group); N: Tumor weights (n = 6 per group). n = 3 independent experiments (n = 4 for Figure 2C), with values expressed as the mean ± SD. Group comparisons were conducted as follows: Independent-samples t-tests or non-parametric tests for two groups; one-way analysis of variance (ANOVA) or non-parametric tests for single-factor multi-group comparisons; and two-way ANOVA for experiments with two independent factors. HCT-116 and SW948 were transfected with short hairpin RNAs targeting EIF5B (sh-EIF5B), which were designated as sh-1, sh-2, and sh-3. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. TCGA: The Cancer Genome Atlas; LV-EIF5B: Lentivirus expressing EIF5B; LV-NC: Lentivirus negative control; NC: Negative control; sh-NC: Short hairpin RNA negative control.

Figure 3 Eukaryotic translation initiation factor 5B drove colonic adenocarcinoma cell migratory and invasive phenotypes. A and B: Transwell migration and invasion assays of eukaryotic translation initiation factor 5B (EIF5B)-overexpressing colonic adenocarcinoma (COAD) cells; C and D: Transwell migration and invasion assays of EIF5B-knockdown COAD cells. All photomicrographs were captured at 200 × magnification. n = 3 independent experiments, with values expressed as the mean ± SD. Independent-samples t-test and non-parametric tests were used for two-group comparisons. ^aP < 0.01. ^bP < 0.001. LV-EIF5B: Lentivirus expressing EIF5B; LV-NC: Lentivirus negative control; NC: Negative control; sh-EIF5B: Short hairpin RNA targeting EIF5B; sh-NC: Short hairpin RNA negative control.

Figure 4 Eukaryotic translation initiation factor 5B depends on the mitogen-activated protein kinase 1 signaling pathway to enhance colonic adenocarcinoma cell proliferation, migration, and invasion. A: Kyoto Encyclopedia of Genes and Genomes enrichment analysis of eukaryotic translation initiation factor 5B (EIF5B) using three independent datasets; B-E: Western blotting was used to investigate the effects of EIF5B overexpression and knockdown on mitogen-activated protein kinase 1 (MAPK1) and phosphorylated MAPK1 expression; F-I: Evaluation of proliferation, migration and invasion in colonic adenocarcinoma (COAD) cells treated as follows: Lentivirus negative control + dimethyl sulfoxide (DMSO), lentivirus expressing EIF5B (LV-EIF5B) + DMSO, and LV-EIF5B + MAPK1 inhibitor SCH772984; J-M: Evaluation of proliferation, migration and invasion in COAD cells treated as follows: Short hairpin RNA negative control + DMSO, short hairpin RNA targeting EIF5B (sh-EIF5B) + DMSO, and sh-EIF5B + MAPK1 activator tert-butylhydroquinone. All photomicrographs were captured at × 200 magnification. n = 3 independent experiments, with values expressed as the mean ± SD. Group comparisons were conducted as follows: Independent-samples t-tests or non-parametric tests for two groups, one-way analysis of variance (ANOVA) or non-parametric tests for single-factor multi-group comparisons and two-way ANOVA for experiments with two independent factors. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. HIV: Human immunodeficiency virus; LV-NC: Lentivirus negative control; MAPK1: Phosphorylated mitogen-activated protein kinase; OD: Optical density; sh-NC: Short hairpin RNA negative control; TCGA: The Cancer Genome Atlas.

Figure 5 Associations of immune infiltration with eukaryotic translation initiation factor 5B expression, along with the underlying signaling pathway. A-C: ESTIMATE algorithm assessed the correlation between eukaryotic translation initiation factor 5B (EIF5B) and immune score; D-F: Correlation of individual genes with immune score was assessed; G: Kyoto Encyclopedia of Genes and Genomes enrichment analyses of immune infiltration. Spearman’s correlation analysis was applied for correlation. COAD: Colonic adenocarcinoma; MAPK: Mitogen-activated protein kinase; TCGA: The Cancer Genome Atlas.

Figure 6 Associations of eukaryotic translation initiation factor 5B expression with drug sensitivity, along with the underlying signaling pathway. A-D: Positive correlations between the sensitivity scores of oxaliplatin 1089, oxaliplatin 1806, 5-fluorouracil 1073, and irinotecan 1088 and EIF5B mRNA expression; E-H: The correlation of individual genes with sensitivity score was analyzed; I: Kyoto Encyclopedia of Genes and Genomes enrichment analyses of sensitivity scores. Spearman’s correlation analysis was applied for correlation. MAPK: Mitogen-activated protein kinase.