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. 2024 Nov 14;14(1):28039.
doi: 10.1038/s41598-024-78663-1.

Tumor suppressor ACER1 correlates with prognosis and Immune Infiltration in head and neck squamous cell carcinoma

Zhixin Liu #  1   2 , Xiaoqi Yang #  2 , Shuai Chen  2 , Wenming Jia  2 , Ye Qian  2 , Minfa Zhang  2   3 , Tianhe Fang  4 , Heng Liu  5 , Hui Yang  6
Affiliations

Tumor suppressor ACER1 correlates with prognosis and Immune Infiltration in head and neck squamous cell carcinoma

Zhixin Liu et al. Sci Rep. .

Abstract

Head and neck squamous cell carcinoma (HNSCC) is notorious for poor prognoses, and effective biomarkers are urgently needed for early diagnosis of HNSCC patients. We investigate the role of alkaline ceramidase 1 (ACER1) and its relationship with immune infiltration in HNSCC. The differential expression and clinical prognostic significance of ACER1 in HNSCC patients are explored using bioinformatics methods and verified in human HNSCC samples. Genetic mutation, DNA methylation and drug sensitivity linked with ACER1 are examined. The potential biological function of ACER1 co-expression genes is assessed, and a series of functional assays are performed on ACER1in vitro. The results comprehensively reveal a relationship between ACER1 and immune infiltration in HNSCC patients. ACER1 expression is significantly downregulated in HNSCC tissues and closely correlated with better prognoses for HNSCC patients, and this prognostic significance is determined by distinct clinical characteristics. Genetic alteration and promoter hypomethylation of ACER1 are involved in progression of HNSCC, and ACER1 expression is significantly related to several drug sensitivities. Functional analysis shows that ACER1 co-expression genes are mainly enriched in the sphingolipid signaling pathway associated with inhibition of tumorigenesis, leading to better prognoses for HNSCC patients. In vitro, ACER1 overexpression inhibits proliferation and migration, induces apoptosis, and promotes adhesion of Fadu and SCC9 cells. In addition, high ACER1 expression is closely linked with infiltration levels of immune cells, and strongly associated with biomarkers of immune cells in HNSCC, suggesting the important role of ACER1 in regulating tumor immunity in HNSCC patients. In summary, ACER1 may be a useful indicator for diagnosis and prognosis, and may regulate immune infiltration in HNSCC patients, thus promising targeted immunotherapy for HNSCC.

Keywords: ACER1; Biomarker; HNSCC; Immune infiltration; Prognosis.

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Conflict of interest statement

Declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate This project was approved by the Institutional Ethics Committee at Qilu Hospital of Shandong University (Ethics approval number: KYLL-2019-2-095). The employment and processing of materials were permitted and obtained informed consent from each patient. All methods involved in human subjects were performed in accordance with the Declaration of Helsinki.

Figures

Fig. 1
Fig. 1
Expression analysis of ACER1 in diverse cancers. (A) Levels of ACER1 expression in multiple types of human cancer using TIMER2.0 database. (B) Differences in ACER1 expression levels between tumorous and adjacent normal tissues of ESCA, HNSCC, SKCM and TGCT, based on the TCGA dataset. (C) ACER1 expression levels in patients at different pathological stages of the four selected cancers. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 2
Fig. 2
Prognostic analysis of ACER1 in different cancers. (A) Overall survival. (B) Disease-free survival for the four tumors detected by ACER1 gene expression using the GEPIA tool.
Fig. 3
Fig. 3
ACER1 expression and its relationship with clinical characteristics in HNSCC patients. (A) Decreased ACER1 expression in HNSCC compared with normal tissues, based on the TCGA database (n = 546). (B) ACER1 mRNA expression analyzed by qRT-PCR in 60 pairs of human HNSCC tissues and normal adjacent tissues. (C) ROC curve analysis of ACER1 in HNSCC. Box-whisker plots reveal ACER1 transcription levels in patients with different subtypes of HNSCC, stratifed according to (D) individual cancer stage, (E) tumor grade, (F) node metastasis status, (G) patient gender, (H) race and (I) age. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 4
Fig. 4
Genetic alteration of ACER1 in HNSCC. (A) Summary of alteration types, corresponding frequencies and mutation sites of ACER1 in the cBioPortal database. (B) ACER1 copy number variations in HNSCC. (C) Survival analysis of ACER1 altered and unaltered groups. (D, E) Mutation types and substitutional mutation types of ACER1 using the COSMIC database.
Fig. 5
Fig. 5
Promoter methylation levels of ACER1 in HNSCC. Box-whisker plots show ACER1 promoter methylation levels in patients with different subtypes of HNSCC, stratified according to (A) sample type, (B) individual cancer stage, (C) tumor grade, (D) node metastasis status, (E) patient gender, (F) tobacco smoking history, (G) TP53 mutation, (H) race and (I) age. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 6
Fig. 6
ACER1 co-expression genes and their functional analysis. (A) Volcano plot of ACER1 co-expression genes. Red dots represent genes positively related to ACER1, and green dots represent negatively related genes. (B) and (C) Top 50 significantly positive and negative genes linked to ACER1 in HNSCC. Functional analysis of ACER1 co-expression genes in HNSCC includes (D) biological processes, (E) cellular components, (F) molecular functions and (G) KEGG pathway using GSEA.
Fig. 7
Fig. 7
ACER1 inhibits growth, induces apoptosis, and regulates cell cycle progression of Fadu and SCC9 cells. (A) Growth curves for Fadu and SCC9 cells transfected with the indicated plasmid, measured by CCK-8 assay. (B) Apoptotic rate of Fadu and SCC9 cells with indicated treatment, measured via flow cytometry from triplicate experiments. (C) Distribution of cell cycle examined by flow cytometry analysis in Fadu and SCC9 cells after overexpressing ACER1.
Fig. 8
Fig. 8
ACER1 suppresses migration, and promotes adhesion of Fadu and SCC9 cells. (A) Wound-healing assay and related analysis after ACER1 overexpression in Fadu and SCC9 cells. (B) Transwell assay and related analysis after ACER1 overexpression in Fadu and SCC9 cells. (C) Cell adhesion assay in Fadu and SCC9 cells after ACER1 overexpression and related analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 9
Fig. 9
Relationship between ACER1 expression and infiltration levels of immune cells in HNSCC using TIMER2.0 database.
Fig. 10
Fig. 10
Relationship between ACER1 expression and biomarkers of immune cells in HNSCC using TIMER2.0 database. Scatterplots of correlations between ACER1 expression and biomarkers of (A) TAM, (B) monocyte, (C) M2 macrophage, (D) dendritic cell and (E) T-cell exhaustion in HNSCC.
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