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

Abstract

BACKGROUND

Although eukaryotic translation initiation factor 5B (EIF5B) is implicated in the pathogenesis of multiple malignancies, its functional impact in colonic adenocarcinoma (COAD) is still not well defined.

AIM

To determine EIF5B’s role, mechanisms, and therapeutic associations in COAD.

METHODS

To assess the prognostic value of EIF5B, we integrated pan-cancer bioinformatic screening with clinical validation in COAD cohorts. The oncogenic function and molecular regulations were evaluated through in vitro and in vivo assays, multi-omic pathway enrichment, Western blotting, rescue experiments, immune infiltration and drug-sensitivity modeling.

RESULTS

Our results showed significant overexpression of EIF5B in COAD tissues, and its correlation with unfavorable patient outcomes. Functional analysis revealed that EIF5B induced migration, proliferation, and invasion in vitro, as well as promoted tumorigenesis in vivo through phosphorylation of mitogen-activated protein kinase 1 (MAPK1). In addition, the immune microenvironment exhibited a suppressed phenotype, accompanied by decreased predictive scores for programmed cell death protein 1/cytotoxic T lymphocyte-associated antigen-4 blockade and reduced predicted sensitivity to chemotherapeutic agents. EIF5B expression, immune evasion, and drug sensitivity were associated with the MAPK signaling pathway.

CONCLUSION

EIF5B mediated phosphorylation of MAPK1 regulates the progression of COAD, suggesting EIF5B as potential therapeutic target for overcoming resistance against chemotherapy and immunotherapy.

Key Words: Colonic adenocarcinoma; Eukaryotic translation initiation factor 5B; Mitogen-activated protein kinase 1; Proliferation; Immune infiltration; Drug sensitivity

Core Tip: This study identified eukaryotic translation initiation factor 5B (EIF5B) as a novel oncogenic driver in colonic adenocarcinoma. EIF5B was upregulated in tumor tissues and associated with poor prognosis. Functional experiments showed that EIF5B promoted tumor cell proliferation, migration, invasion, and xenograft growth by enhancing mitogen-activated protein kinase 1 phosphorylation. High EIF5B expression was also linked to an immunosuppressive tumor microenvironment, lower predicted responses to programmed cell death protein 1/cytotoxic T lymphocyte-associated antigen-4 blockade, and reduced sensitivity to standard chemotherapeutic agents. EIF5B may serve as both a prognostic biomarker and therapeutic target for overcoming chemotherapy and immunotherapy resistance in colonic adenocarcinoma.

INTRODUCTION

Colorectal cancer represents a global health crisis, accounting for approximately 10% of all new cancer diagnoses. Pathological diagnoses have revealed that colonic adenocarcinoma (COAD) is the most prominent subtype as indicated by pathological diagnosis[1]. Furthermore, it ranks as the fourth leading cause of global cancer-related mortality[2]. Earlier data based on epidemiological trends have indicated that COAD incidence has been increased among individuals under the age of 50[3,4]. To overcome challenges associated with COAD due to high incidence and poor prognosis[5], it is necessary to elucidate the molecular mechanisms underlying COAD pathogenesis to develop advanced and effective therapeutic strategies.

Eukaryotic translation initiation factors play a pivotal role in the assembly of the translation initiation complex as they primarily regulate the orchestration of protein synthesis in eukaryotic cells[6]. Eukaryotic translation initiation factor 5B (EIF5B) ensures the stable positioning of the initiator methionyl-tRNA within the ribosome by facilitating the junction of the ribosomal subunits[7,8]. EIF5B is a widely conserved eukaryotic protein encoded by the EIF5B gene[9,10]. Earlier reports have indicated that the altered expression of EIF5B induces cancer initiation and progression[11,12]. For instance, in the case of glioblastoma, EIF5B enhances the translation of mRNA containing specific sites of ribosomal entry, thereby contributing to tumor progression[11,13]. In the case of hepatocellular carcinoma, overexpressed EIF5B can induce the development of cancer[14], while in prostate cancer, it drives malignant phenotypes such as proliferation and invasion[6]. Although researchers have reported the association of EIF5B with cancer, however, its functional significance is yet to be explored in COAD.

Therapeutic or chemical resistance is one of the most important challenges faced by clinicians or oncologists. In this aspect, tumor microenvironment is important to consider, which contains immune cells, endothelial cells, and other regulatory molecules[15]. The infiltrating immune cells determine the therapeutic efficacy, as well as advancement of cancer[16]; thus immunotherapy is considered one of the promising therapeutic options available for cancer[17]. However, these therapeutic strategies face resistance as a major challenging barrier, which needs attention.

Therefore, in this study, we present EIF5B as an important biomarker that can be considered for the development of an efficient therapeutic strategy. We explored the correlation between EIF5B and patient prognosis and investigated the effects of EIF5B modulation on key cellular behaviors of COAD cells, including proliferation, migration, and invasion. Furthermore, we explored the regulatory role of EIF5B in tumor infiltration and its influence on therapeutic efficacy. Thus, this study identified EIF5B as a suitable therapeutic target and potential biomarker for personalized intervention in COAD.

MATERIALS AND METHODS

Patients and tissue samples

Seventy pairs of tissue specimens were collected from patients with COAD and confirmed through histopathological examination. Immunohistochemical staining was performed on 70 COAD tissues along with their corresponding normal colonic tissues. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan Province, China; No. KY2025211 and dated June 3, 2025). Informed consent was obtained from all subjects involved in the study. The relevant clinical characteristics of the patient cohort are presented in Table 1.

Table 1 Clinical characteristics of patients with colonic adenocarcinoma from colonic adenocarcinoma clinical specimens, n (%).

Characteristics
Tumor location
Right	40 (57.1)
Left	30 (42.9)
Age (years)
≤ 65	35 (50.0)
> 65	35 (50.0)
Sex
Male	37 (52.9)
Female	33 (47.1)
T stage
T1	5 (7.1)
T2	10 (14.3)
T3	45 (64.3)
T4	10 (14.3)
N stage
N0	41 (58.6)
N1	21 (30.0)
N2	8 (11.4)
M stage
M0	59 (84.3)
M1	11 (15.7)
Grade
Well differentiated	13 (18.6)
Moderately differentiated	45 (64.3)
Poorly differentiated	12 (17.1)
Overall survival
Alive	48 (68.6)
Dead	22 (31.4)

Patient characteristics

Inclusion criteria comprised patients with a confirmed pathological diagnosis of COAD and complete clinicopathological data. Exclusion criteria included samples lacking essential pathological information, patients with a history of other malignancies, and those who had received prior radiotherapy or chemotherapy. All cases were consecutively collected from January 2022 to December 2023 at the Department of General Surgery, Second Affiliated Hospital of Zhengzhou University, and no additional subjective selection or exclusion was applied beyond the predefined criteria.

The Cancer Genome Atlas and Gene Expression Omnibus data collection and processing

The COAD dataset from The Cancer Genome Atlas (TCGA) (https://xena.ucsc.edu/public/)[18] was applied to analyze EIF5B’s prognostic value. The relevant datasets in the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/)[19-23] to evaluate EIF5B expression.

Immunohistochemistry assay

For immunohistochemistry staining, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Following antigen retrieval with citrate buffer, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide. Sections were then incubated overnight with a primary antibody targeting EIF5B (1:250, 13527-1-AP; Proteintech, Wuhan, China), washed in phosphate-buffered saline (PBS), and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (RGAR011; Proteintech). After additional PBS washes, immunoreactivity was visualized using 3,3’-diaminobenzidine as the chromogen, and nuclei were counterstained with hematoxylin.

EIF5B expression was quantified using a semi-quantitative immunoreactivity scoring system, integrating both staining intensity (scored as 0: Negative, 1: Weak, 2: Moderate, 3: Strong) and the percentage of positive cells (scored as 0: 0%, 1: ≤ 10%, 2: 11%-50%, 3: 51%-80%, 4: ≥ 80%). Two pathologists, blinded to patient information, independently assessed the immunohistochemical staining.

Cell culture and lentiviral infection

The human COAD cell lines CW2, RKO, HCT-116, and SW948 were cultured under standard conditions (37 °C, 5% carbon dioxide, humidified atmosphere). CW2 and RKO cells were infected with lentiviral particles encoding EIF5B (GeneChem Co., Ltd., Shanghai, China) based on the multiplicity of infection value. HCT-116 and SW948 were transfected with short hairpin RNAs targeting EIF5B, which were designated as sh-1, sh-2, and sh-3. Lentivirus-transfected cells were selected with puromycin. Cells with empty vector transfection served as negative controls.

Quantitative PCR

Total RNA was extracted from COAD cell lines using TRIzol (Invitrogen, Carlsbad, CA, United States). Reverse transcription was carried out with a commercial kit (Vazyme, Nanjing, Jiangsu Province, China) to generate complementary DNA. Amplification was performed with the following primer sets: EIF5B (forward: 5’-AGGGCTTATGACAAAGCAAAACG-3’, reverse: 5’-CCCAAGTACGCAGATAATAGGGG-3’) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’, reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’). Relative expression was determined by the 2^-ΔΔCt method, normalizing to GAPDH as the endogenous control.

Western blotting

Total cellular proteins were lysed in radio immunoprecipitation assay buffer (Cwbio, Jiangsu Province, China) and protein concentrations were determined with a bicinchoninic acid assay kit (Beyotime, Shanghai, China). Protein samples (30 μg/Lane) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes. After blocking in 5% skim milk for 2 hours at room temperature, the membranes were incubated with primary antibodies overnight, followed by an HRP-conjugated secondary antibody (SA00001-2; Proteintech). Protein signals were detected using an enhanced chemiluminescence reagent kit (Abbkine Scientific Co., Ltd., Wuhan, Hubei, China). Primary antibodies used were: EIF5B (13527-1-AP; Proteintech), phosphorylated mitogen-activated protein kinase 1 (MAPK1) (AP0485; Abclonal, China), MAPK1 (A0229; Abclonal), and β-actin (AC026; Abclonal).

In vitro assays

Cell proliferation ability was evaluated with the Cell Counting Kit-8 (CCK-8) assay (Abbkine Scientific Co., Ltd.). Cells were plated in 96-well plates. The absorbance at 450 nm was measured at 24 hours, 48 hours, 72 hours, and 96 hours after the addition of CCK-8 reagent. For cloning capability, cells were plated in 6-well plates and cultured for 15 days. The resulting colonies were fixed and stained for quantification.

Cell migration and invasion abilities were assessed using Transwell plates. In the migration assay, cells suspended in serum-free medium were placed in the upper chamber, with the lower chamber filled with 600 μL of complete medium containing 10% fetal bovine serum. For the invasion assay, matrigel was pre-laid in the upper. Following 48 hours of incubation, cells that had traversed the membrane were fixed, stained, and counted under a microscope.

Tumor xenograft implantation in nude mice

All animal procedures were performed following the Guidelines for Animal Care and were approved by the Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (No. KY2025211 and dated June 3rd, 2025). Four- to six-week-old nude mice were housed under specific pathogen free conditions. For the tumor-bearing model, 2 × 10⁶ cells were subcutaneously injected into each mouse. Tumor dimensions were recorded every five days, and volume was computed as 1/2 × (largest diameter) × (smallest diameter)².

Immune infiltration analysis

Immune infiltration within COAD samples was evaluated using “ESTIMATE algorithm and single-sample gene set enrichment analysis (ssGSEA).” ssGSEA was conducted based on immune gene sets[24] via R (v4.1.2), utilizing the GSVA (v1.42.0), Biobase (v2.54.0), genefilter (v1.76.0), and stringr (v1.5.1) packages. ESTIMATE analysis was performed based on “gene.gct” and “estimate-score.gct” by using estimate(v1.0.13) package in the R (v4.1.2).

Calculation of the scores for immune checkpoint inhibitors therapy

The immunophenoscore (IPS), a well-validated biomarker that predicts response to immune checkpoint inhibitors (ICIs) therapy, was acquired from The Cancer Immunome Atlas (TCIA) (https://tcia.at/patients). Four types of IPS were calculated: IPS-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-negative-programmed cell death protein 1 (PD-1)-negative, IPS-CTLA-4-negative-PD-1-positive, IPS-CTLA-4-positive-PD-1-negative, and IPS-CTLA-4-positive-PD-1-positive. These subtypes correspond to predicted efficacy under the following therapeutic conditions: No blockade, anti-PD-1 monotherapy, anti-CTLA-4 monotherapy, and combined anti-CTLA-4/anti-PD-1 blockade, respectively.

Evaluation of drug sensitivity

Drug sensitivity for the TCGA-COAD dataset was predicted using oncoPredict (v1.2) in R (v4.1.2). Ridge regression models trained on GDSC2 data (GDSC2 Expr.rds and GDSC2 Res.rds) were applied to the TCGA-COAD dataset. Higher sensitivity scores indicated lower drug sensitivity.

Bioinformatics based tools and analysis

Spearman’s correlation analysis was performed using R (v4.1.2). Genes exhibiting significant correlations (r ≥ 0.30, P < 0.05) with EIF5B expression, immune infiltration scores, or drug sensitivity were identified. Subsequently, three distinct gene sets were independently analyzed for Gene Ontology-Biological Process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment via the Metascape platform (http://metascape.org/). Multiple-testing corrections were consistently applied across all enrichment and correlation analyses.

Statistical analyses

Data are presented as the mean ± SDs of three independent experiments. Group comparisons employed the Student’s t-test (paired or unpaired) or one-way analysis of variance (ANOVA) for normally distributed data; two-way ANOVA was used for analyses involving multiple variables. Normality was assessed using the Shapiro-Wilk test for n < 50 and Kolmogorov-Smirnov test for n ≥ 50. Non-parametric tests were applied when data violated assumptions of normality or homogeneity of variance. For immunohistochemistry H-score cutoffs, we used X tile to determine the optimal cutoff in Cox regression and survival analysis. For the correlation analysis between clinical characteristics of patients with COAD and EIF5B expression, we divided patients into two groups based on the H-score: Low expression (H-score ≤ 6, 34 patients) and high expression (H-score > 6, 36 patients). Survival analysis was evaluated by Kaplan-Meier curves and the Log-Rank test. Prognostic factors were identified using univariate and multivariate Cox regression, with variables showing P < 0.2 in univariate analysis entered into the multivariate model. Correlations were assessed with Pearson’s or Spearman’s coefficients based on data distribution. Categorical variables are presented as n (%) and compared by the χ² test. Statistical analyses were conducted with SPSS 26.0 software (IBM, Armonk, NY, United States) and GraphPad Prism 8.0.2 software (GraphPad Software, La Jolla, CA, United States). P < 0.05 was considered statistically significant.

RESULTS

Upregulation of EIF5B in COAD correlates with malignant progression and poor clinical outcome

Integrated analysis of transcriptomic data from the TCGA-COAD and GEO datasets demonstrated that EIF5B mRNA expression was significantly upregulated, whereas immunohistochemistry analysis depicted higher expression of EIF5B protein in COAD tissues compared with normal tissues (Figure 1A-G). In addition, quantitative analysis showed a significant enrichment of EIF5B overexpression in COAD samples, which worsens the overall survival in patients (Figure 1H). Tumor location, its advanced stages, and elevated EIF5B were determined as prognostic factors by univariate Cox analysis, while multivariate Cox analysis showed that N-stage and elevated EIF5B were potential independent poor prognostic factors. Correlation analysis showed that T-stage of cancer is directly associated with expression levels of EIF5B (Tables 2 and 3). As depicted in Supplementary Figure 1, EIF5B linked genes contribute to microtubule processes and cellular proliferation.

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.

Table 2 Cox regression analysis outcomes for clinical characteristics of colonic adenocarcinoma patients and eukaryotic translation initiation factor 5B expression.

Variable		Total (n = 70)	Univariate Cox analysis
			Hazard ratio	95%CI	P value
Tumor location	Right	40
	Left	30	0.408	0.158-1.054	0.064
Age	≤ 65	35
	> 65	35	1.462	0.631-3.388	0.375
Sex	Male	37
	Female	33	1.408	0.608-3.261	0.424
T stage	T1 + T2	15
	T3 + T4	55	7.952	1.638-38.590	0.010
N stage	N0	41
	N1 + N2	29	12.276	3.610-41.747	0.000
M stage	M0	59
	M1	11	3.898	1.535-9.898	0.004
Grade	Well differentiated	13			0.047
	Moderately differentiated	45	4.677	0.617-35.447	0.136
	Poorly differentiated	12	11.287	1.334-95.476	0.026
EIF5B	High (reference)	44
	Low	26	0.203	0.060-0.692	0.011
			Multivariate Cox analysis
Tumor location	Right	40
	Left	30	0.399	0.126-1.263	0.118
T stage	T1 + T2	15
	T3 + T4	55	1.962	0.224-17.189	0.543
N stage	N0	41
	N1 + N2	29	14.023	2.972-66.612	0.001
M stage	M0	59
	M1	11	1.489	0.506-4.389	0.470
Grade	Well differentiated	13			0.888
	Moderately differentiated	45	1.654	0.200-13.680	0.640
	Poorly differentiated	12	1.518	0.147-15.687	0.726
EIF5B	High (reference)	44
	Low	26	0.120	0.029-0.487	0.003

CI: Confidence interval; EIF5B: Eukaryotic translation initiation factor 5B.

Table 3 The correlation between clinical characteristics of colonic adenocarcinoma patients and eukaryotic translation initiation factor 5B expression.

Characteristics	n = 70	EIF5B		χ2 value	P value
		Low	High
T stage				4.686	0.030
T1 + T2	15	11	4
T3 + T4	55	23	32
N stage				0.278	0.598
N0	41	21	20
N1 + N2	29	13	16
M stage				0.051	0.822
M0	59	29	30
M1	11	5	6

EIF5B promotes COAD cell proliferation and malignant progression

To demonstrate the biological role of EIF5B in COAD, the genes correlated with EIF5B expression were identified based on the correlation coefficient and P value (Figure 2A). Enrichment analyses of GO-BP indicated that EIF5B-associated genes were predominantly involved in pathways associated with cell proliferation biological processes associated with microtubules (Figure 2B), suggesting a potential role of EIF5B in tumor growth. Next, EIF5B expression in the COAD cell line was quantified using quantitative PCR (qPCR) to select cell lines for subsequent experiments (Figure 2C). Using lentiviral transfection, we constructed stable EIF5B-overexpressing and EIF5B-knockdown cell lines as well as their corresponding control cell lines. Validation by qPCR and confirmed successful EIF5B overexpression in the LV-EIF5B cell lines (Figure 2D and E) and downregulated in sh-EIF5B cell lines (Figure 2F and G) compared with controls. Among the tested shRNAs, sh-2 exhibited highest knockdown efficiency, and was therefore selected for follow-up experiments. These findings confirm the successful establishment of EIF5B-overexpressing and EIF5B-knockdown COAD cell models, suitable for subsequent functional studies. To functionally assess EIF5B’s role in COAD cell proliferation, CCK-8 and colony formation assays were performed. The overexpression of EIF5B significantly enhanced, while its knockdown suppressed cell proliferation in COAD (Figure 2H-K). The tumor-promoting effect of EIF5B was further assessed in vivo using a nude mouse xenograft model using stable RKO cell lines. As shown in Figure 2L, xenografts derived from EIF5B-overexpressing RKO cells showed significantly accelerated tumor growth compared with xenografts derived from negative control cells. Consistently, on day 33 after inoculation, the xenografts from EIF5B-overexpressing cells displayed significantly increased tumor volume and weight than those from negative control RKO cells in mice (Figure 2M and N).

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.

EIF5B induces progression of COAD by activating MAPK1 signaling

The effects of EIF5B on the metastatic potential of COAD cells were tested Transwell migration and invasion assays. The obtained results showed that EIF5B overexpression significantly increased migration and invasion potential of CW2 and RKO cells (Figure 3A and B). In contrast, EIF5B knockdown substantially suppressed migration and invasion capabilities of HCT-116 and SW948 cells (Figure 3C and D). KEGG enrichment analyses indicated that the MAPK pathway ranked within the top 10 in all three datasets (TCGA, GSE39582, and GSE41258) and its consistent enrichment across all three datasets strongly supports its relevance (Figure 4A). Given that MAPK1 has been identified as a downstream target of EIF5B[25], we examined the effect of EIF5B on the expression of MAPK1 and its phosphorylated form. Western blotting analysis showed significant increase in phosphorylation of MAPK1 levels in the case of overexpressed EIF5B (Figure 4B and C). On the other hand, EIF5B knockdown mitigated the phosphorylation of MAPK1 (Figure 4D and E). These results indicate that phosphorylated MAPK1 induces the oncogenic potential of EIF5B as confirmed by rescue experiments performed through pharmacological inhibition of MAPK1 signaling. The successful inhibition reduced the potential of EIF5B to cause cell proliferation and migration (Figure 4F-I). On the other hand, activated MAPK1 signaling in response to activation overexpression of EIF5B restored its capability to induce cell proliferation and migration (Figure 4J-M). Overall, these results indicate that EIF5B mediated activation of MAPK1 induces the progression of COAD by inducing cell proliferation, invasion and migration.

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.

EIF5B is associated with tumor microenvironment in the aspects of immunosuppression

To investigate the relationship between EIF5B expression and the tumor immune microenvironment in COAD, we employed the ESTIMATE and ssGSEA methods across three independent datasets (TCGA-COAD, GSE39582, GSE41258). Correlation analysis demonstrated an inverse relationship between EIF5B expression and ESTIMATE-derived immune scores (Figure 5A-C). Consistently, ssGSEA analysis demonstrated that elevated EIF5B expression was negatively correlated with infiltration levels of multiple immune cell subsets, including activated B cells, macrophages, effector memory cluster of differentiation 8⁺ T cells, and several helper T cell populations (Supplementary Table 1). Additionally, we analyzed the relationship between EIF5B expression and ICIs therapy scores using the TCIA, showing that high EIF5B expression correlated with lower treatment response scores for PD-1/CTLA-4 inhibitors (Table 4). To identify the signaling pathway underlying the immune infiltration, the genes correlated with immune score were selected according to the correlation coefficient and P value (Figure 5D-F). Our findings suggested a potential link between MAPK signaling pathway and immune infiltration within the COAD microenvironment (Figure 5G). Overall, these findings imply that EIF5B levels may affect COAD immune infiltration via MAPK signaling pathway.

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.

Table 4 Association between eukaryotic translation initiation factor 5B and immunophenoscore scores using The Cancer Immunome Atlas database.

Characteristics	r value	P value
IPS-CTLA-4-negative-PD-1-negative1	-0.128	0.019
IPS-CTLA-4-negative-PD-1-positive2	-0.137	0.012
IPS-CTLA-4-positive-PD-1-negative3	-0.172	0.002
IPS-CTLA-4-positive-PD-1-positive4	-0.189	< 0.001

¹Predicted efficacy under no anti-cytotoxic T lymphocyte-associated antigen-4/anti-programmed cell death protein 1 blockade.

²Predicted efficacy under anti-programmed cell death protein 1 monotherapy.

³Predicted efficacy under anti-cytotoxic T lymphocyte-associated antigen-4 monotherapy.

⁴Predicted efficacy under combined anti-cytotoxic T lymphocyte-associated antigen-4/anti-programmed cell death protein 1 blockade.

CTLA-4: Cytotoxic T lymphocyte-associated antigen-4; IPS: Immunophenoscore; PD-1: Programmed cell death protein 1.

EIF5B expression is associated with chemotherapy sensitivity in COAD

Given that chemotherapy remains a cornerstone of COAD treatment, we investigated the responses of EIF5B expression to common chemotherapeutic agents. Correlation analysis demonstrated that sensitivity scores for oxaliplatin 1089, oxaliplatin 1806, 5-fluorouracil 1073, and irinotecan 1088 were positively correlated with EIF5B levels (Figure 6A-D). To identify the signaling pathway underlying drug sensitivity, the genes correlated with drug sensitivity were selected according to the correlation coefficient and P value (Figure 6E-H). The enrichment analyses indicated that the MAPK signaling pathway may correlate with the sensitivity of COAD to oxaliplatin, 5-fluorouracil and irinotecan (Figure 6I). Given the higher sensitivity scores corresponded to reduced chemotherapeutic responsiveness, these findings suggest that overexpression of EIF5B may be associated with chemoresistance in COAD, potentially through dysregulation of MAPK signaling. Overall, these results indicate EIF5B as potential biomarker to predict chemotherapeutic response as well as a candidate target to overcome drug resistance in COAD.

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.

DISCUSSION

Cellular translation serves as a pivotal regulatory node in gene expression, and its dysregulation is a hallmark of oncogenic transformation. Numerous studies indicate that eukaryotic initiation factor family members are involved in regulating malignant cellular behaviors, and their aberrant expression in various human cancers has been implicated in driving tumorigenic processes[26]. Transforming growth factor-β signaling enhances EIF5A2 expression, thereby promoting cell migration, while EIF3A is directly associated with tumor growth[27]. It is evident that EIF5B is associated with cancer, however, its functional role in COAD pathogenesis has not been reported previously. Therefore, we believe that this study is the first to discuss the oncogenic role of EIF5B in COAD. Our analysis based on bioinformatics and immunohistochemistry revealed a marked upregulation of EIF5B in COAD tissues, while survival analysis showed that high EIF5B expression is associated with poor overall survival. Our findings suggest that high EIF5B expression may be an independent risk factor for COAD patients, however, the relatively small sample size necessitates further validation in larger cohorts. Furthermore, GO-BP enrichment analysis indicated that EIF5B is associated with regulation of cell proliferation and microtubule functions, which are vital for cell migration and proliferation[28]. Earlier reports have shown that EIF3B, EIF4A3 and EIF5A2 promote cell proliferation in lung adenocarcinoma, and other associated cancers[29-31]. In the agreement of these reports, we found that EIF5B induces cell proliferation, thereby accelerating tumor growth. Overall, our results confirm that EIF5B expression is directly associated with the progression and development of COAD progression. We know that MAPK signaling pathway is associated with cell survival and immune responses[32]. Similarly, previous studies have reported that MAPK pathway-related proteins were downregulated in EIF5B-knockdown 293T cells[25]. Therefore, we intended to investigate this association. Results obtained from western blotting analysis indicated that EIF5B failed to promote the expression of MAPK1, however, phosphorylation level of MAPK1 was significantly enhanced. This further confirmed the previous indication that MAPK1 phosphorylation is a noticeable player in development of COAD and its metastasis[33].

To understand the association of EIF5B with cell proliferation and invasion, we inhibited MAPK1 using SCH772984, which mitigated the potential of EIF5B to induce cell invasion, progression and migration. On the other hand, activating MAPK1 using tert-butylhydroquinone (TBHQ) reversed function, suggesting that EIF5B promotes cancer progression by activating MAPK1 signaling pathway.

We know that immunotherapy and chemotherapy are the prominent treatment strategies used in cancer management, which face challenges such as therapeutic resistance. Therefore, we further elucidated the role of EIF5B in this therapeutic resistance. Our analysis based on ESTIMATE algorithm showed a negative correlation between EIF5B levels and immune scores such that overexpressed EIF5B reduced infiltration of immune cells. Moreover, our analysis showed that the expression of EIF5B is inversely related to the predicted treatment response scores for inhibitors. Among these indicators, we noted that MAPK signaling pathway was a key player in immune infiltration in COAD, which performs critical role in immune response regulation[34]. Considering the aforementioned indications, EIF5B could influence immune infiltration through phosphorylating MAPK signaling pathway, therefore, targeting EIF5B can be considered as a promising therapeutic option.

Considering the therapeutic options, we note that EIF5B overexpression reduces responsiveness to 5-fluorouracil, oxaliplatin, and irinotecan-based therapies in COAD. Consistent with our current results, earlier studies have reported that MAPK signaling is predominantly associated with resistance in response to 5-fluorouracil, oxaliplatin, and irinotecan[34,35], suggesting that EIF5B can affect the responsiveness of chemotherapy in COAD.

In this study, we report that EIF5B mediated phosphorylation is important in the development and progression of cancer as well as the responsiveness to therapeutic strategies. However, further verification of MAPK1 signaling pathway on tumor development is required using nude mice, as well as the effect of EIF5B knockdown on tumor growth should also be verified. The precise molecular mechanism by which EIF5B affects MAPK1 expression or phosphorylation requires further experimental validation. Additionally, low-magnification photographs of the Transwell assays were not captured, the relatively small number of clinical samples necessitates increasing the sample size, and the MAPK1 activator TBHQ exhibits limited specificity. This study is also limited by validation of correlations between EIF5B and therapy scores both in vitro and in vivo experiments. Therefore, future experiments should also focus on directly elucidating the impact of EIF5B on immune cell recruitment and therapy response using relevant animal models or treated models. Some of the important results presented in this study are based on computational analysis, especially the influence of EIF5B on chemosensitivity. Thus, further experiments should consider confirming these results by utilizing cell lines as well as animal models as appropriate. Additionally, the immune and drug sensitivity pathway analyses rely on correlative data and in silico predictions, requiring direct experimental validation. Lastly, the influence of EIF5B on tumor metastasis should be investigated using a liver metastasis model in nude mice.

CONCLUSION

Our findings demonstrate that elevated EIF5B expression is a critical factor in the carcinogenesis and progression of COAD, while simultaneously contributing to resistance against ICIs therapy and conventional chemotherapy. EIF5B drives tumor cell proliferation, migration and invasion by activating the MAPK1 signaling pathway. This work identifies EIF5B as a pivotal driver of malignancy and highlights its clinical value as a dual-purpose target for both prognostic stratification and therapeutic intervention in COAD.