Low expression and Hypermethylation of ATP2B1 in Intrahepatic Cholangiocarcinoma Correlated With Cold Tumor Microenvironment

Abstract

Background: The efficacy of current therapeutic schedule is limited owing to fibroproliferative tumor microenvironment (TME) of cholangiocarcinoma, compelling a search for new therapeutic targets.

Methods: Gene expression profiles and methylation profiles were obtained from UCSC Xena. Consensus clustering was performed on the transcriptome data of cholangiocarcinoma to determine the different immune subtypes. The differentially expressed genes (DEGs) between hot tumor and cold tumors were identified. ESTIMATE was used to assess immune score, and the cases were separated into relatively superior and inferior immune score groups. Single-sample gene set enrichment analysis was applied to assess 28 immune cells in the cholangiocarcinoma microenvironment. Unsupervised consensus was applied for methylation profiling to distribute the high and low methylation groups. The correlation between DNA methylation and mRNA expression was investigated, and the relationship between the ATP2B1 gene and the immune microenvironment was explored. Finally, 77 cases of intrahepatic cholangiocarcinoma (ICC) were collected for verification.

Results: Seven subtypes were related to patient outcomes (P=0.005). The proportions of CD8⁺ T cells in the "hot" immune type was significantly greater than that in the "cold" immune type (P<0.05). Next, DEGs and DNA methylation-governed genes were intersected, and ATP2B1 was identified as a prognosis factor in ICC (P=0.035). ATP2B1 expression was positively correlated with immune scores (P=0.005, r=0.458), the levels of infiltrating CD8⁺ T cells (P=0.004, r=0.47), and CD4⁺ T cells (P=0.027, r=0.37). Immunohistochemistry confirmed that the amounts of CD8⁺ and CD4⁺ T cells were significantly higher in ICC tissue samples than in tissues with ATP2B1 overexpression (P<0.05).

Conclusions: ATP2B1 overexpression can activate immune signals and prompt cold tumor response.

Keywords: ATP2B1; Ca2+; immune subtypes; intrahepatic cholangiocarcinoma; treatment.

Figure 1

Immunological characterization of cholangiocarcinoma has been implicated in overall survival. (A) Heatmap of the immune clusters of cholangiocarcinoma; (B) Immune score, stromal score, and tumor purity in the seven clusters; (C) Differential immune cells between cold and hot tumors; (D) Kaplan–Meier curves of the association of the seven subtypes with overall survival.

Figure 2

DNA methylation-regulated tumor immunity in cholangiocarcinoma. (A) Kaplan–Meier curves of the association of DNA methylation with overall survival; (B) Correlation between transcriptome and methylation; (C) Differences of 28 immune cells between hypermethylation and hypomethylation.

Figure 3

Prognostic significance of ATP2B1 in ICC. (A) Pathway enrichment analyses and semantic similarities of gene classes by GOSemSim; (B) Expression of four genes in cholangiocarcinoma from TCGA; (C) Kaplan–Meier curves of the association of ATP2B1 gene methylation with overall survival; (D) Representative IHC staining of ATP2B1 in ICC tissues; (E) Proportions of ICC case with different ATP2B1 expression levels; (F) Association of ATP2B1 expression with better disease-free survival in ICC.

Figure 4

ATP2B1 remodeling the tumor immune microenvironment. (A) Kaplan–Meier curves of the correlation of immune score with the prognosis of overall survival; (B) Correlation between ATP2B1 expression and the tumor immune microenvironment; (C) Differences of 28 immune cells in the high and low ATP2B1 expression groups.

Figure 5

Association of CD8+T cells, CD4+T cells (A), neutrophil (B), macrophage and myeloid dendritic cells (C) with ATP2B1 expression.

Figure 6

ATP2B1 expression in CD4⁺ and CD8⁺ immune cells. (A) IHC staining analysis of CD4⁺ and CD8⁺ immune cells; (B) Numbers of CD4⁺ and CD8⁺cells detected by IHC. *P < 0.05; **P < 0.01; ***P < 0.001.