ADH4-a potential prognostic marker for hepatocellular carcinoma with possible immune-related implications

Abstract

Objective: This study aims to explore ADH4 expression in hepatocellular carcinoma (HCC), its prognostic impact, and its immune correlation to provide novel insights into HCC prognostication and treatment.

Methods: HCC prognostic marker genes were rigorously selected using GEO database, Lasso regression, GEPIA, Kaplan-Meier and pROC analyses. The expression of interested markers (ADH4, DNASE1L3, RDH16, LCAT, HGFAC) in HCC and adjacent tissues was assessed by Immunohistochemistry (IHC). We observed that ADH4 exhibited low expression levels in liver cancer tissues and high expression levels in normal liver tissues. However, the remaining four genes did not manifest any statistically significant differences between hepatocellular carcinoma (HCC) tissue and adjacent non-cancerous tissue. Consequently, ADH4 became the primary focus of our research. ADH4 expression was validated by signed-rank tests and unpaired Wilcoxon rank sum tests across pan-cancer and HCC datasets. Clinical significance and associations with clinicopathological variables were determined using Kaplan-Meier, logistic regression and Cox analyses on TCGA data. The ADH4-related immune responses were explored by Spearman correlation analysis using TIMER2 data. CD68, CD4, and CD19 protein levels were confirmed by IHC in HCC and non-cancerous tissues.

Results: ADH4 showed significant downregulation in various cancers, particularly in HCC. Moreover, low ADH4 expression was associated with clinicopathological variables and served as an independent prognostic marker for HCC patients. Additionally, ADH4 affects a variety of biochemical functions and may influence cancer development, prognosis, and treatment by binding to immune cells. Furthermore, at the immune level, the low expression pattern of ADH4 is TME-specific, indicating that ADH4 has the potential to be used as a target for cancer immunotherapy.

Conclusion: This study highlights the diagnostic, prognostic and immunomodulatory roles of ADH4 in HCC. ADH4 could serve as a valuable biomarker for HCC diagnosis and prognosis, as well as a potential target for immunotherapeutic interventions.

Keywords: Alcohol dehydrogenase 4 (ADH4); Hepatocellular carcinoma; Immune-related.

Fig. 1

The differential gene analysis and screening of prognostic marker genes in HCC. (A-D) The volcano plot of the four data sets. (E) The DEG intersection in a Venn diagram. (F) The LASSO model-related ten-time cross-validation for tuning parameter selection. (G) The profiles of LASSO coefficients. (H) The risk scores, survival statuses, and heat map of the five genes in patients with HCC

Fig. 2

The expression, survival, and ROC curve analysis of the hub genes. (A-E) The levels of the 5 hub genes in LIHC and normal samples (Tumor Color: red; Normal Color: gray). (F-J) The levels of the five hub genes in paired LIHC samples. (K-O) Survival analyses for the five hub genes. (P-T) ROC curve analyses for the five hub genes

Fig. 3

Analysis of ADH4 Expression in Pan-Cancer and Hepatocellular Carcinoma. (A) Expression levels of ADH4 across different cancer types. (B) Analysis of ADH4 expression in matched pairs of tumor and normal tissues. (C-D) ADH4 mRNA and protein expression in hepatocellular carcinoma according to the UALCAN database. (E) Representative immunohistochemical images of ADH4 expression in liver cancer tissues and normal controls. (F) Statistical analysis of mean staining intensity of immunohistochemical images from 30 patients with liver cancer and their adjacent tissues. *p < 0.05, * *p < 0.01, * **p < 0.001, ** **p < 0.0001, ns: not statistically significant

Fig. 4

Association between ADH4 mRNA Expression and Clinical Parameters in HCC. (A) Comparison of ADH4 mRNA expression between HCC tissues and normal controls. (B-R) The ADH4 mRNA expression in HCC based on analysis of the Cancer Genome Atlas (TCGA) using R. Parameters investigated include N stage, T stage, M stage, tumor status, gender, age, weight, height, BMI, AFP (ng/ml), albumin (g/dl), vascular invasion, pathologic stage, adjacent hepatic tissue inflammation, race, Fibrosis Ishak score, and histologic grade. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

The prognostic analysis of ADH4 in HCC. (A-D) Analysis results for Overall Survival (OS), Recurrence-Free Survival (RFS), Disease-Specific Survival (DSS), and Progression-Free Survival (PFS), respectively. (E) The Nomogram for predicting 1-year, 3-year, and 5-year OS probabilities for patients with HCC. (F) The Nomogram Calibration Plots for predicting 1, 3, and 5-year OS probabilities. *(Note OS = overall survival, RFS = recurrence-free survival, PFS = progression-free survival, DSS = disease-specific survival, HCC = hepatocellular carcinoma, ADH4 = alcohol dehydrogenase 4)*

Fig. 6

The analysis of immune cell infiltration in relation to ADH4 expression is presented in this figure. (A) Forest plot that depicts the correlation between the level of immune cells and ADH4 mRNA expression. (B) The contrast in the abundance levels of neutrophils, Tcm, Th2, NK CD56bright, and TFH cells between high and low ADH4 expression groups. (C) The correlation between the expression level of ADH4 and immune cell infiltration in hepatocellular carcinoma, focusing on neutrophils, Tcm, Th2, NK CD56bright, and TFH cells

Fig. 7

ADH4 negatively correlated with tumor-associated macrophage (TAM) infiltration in hepatocellular carcinoma (HCC) tissues. (A and B) Representative immunohistochemical (IHC) staining images of ADH4 and CD68 in HCC tissues and in adjacent normal tissues. Scale bar: 40 μm

Fig. 8

The correlation between ADH4 expression and immune checkpoint proteins was investigated in liver hepatocellular carcinoma (LIHC). (A) Correlation between ADH4 and immune checkpoints molecules in pan-cancers. (B) Scatter diagrams demonstrated a negative correlation between ADH4 mRNA expression and the expression of these proteins. (C) A heatmap of co-expression between ADH4 and the proteins VEGFB, CD276, TGFB1, TNFRSF4, TNFRSF18, TNFSF9, PDCD1, and CTLA4. (D) In the group with low ADH4 expression, VEGFB, CD276, TGFB1, and TNFRSF4 proteins were significantly upregulated compared to the group with high ADH4 expression. (***P < 0.001)