Hyper-methylation and DNMT3A mediated LTC4S downregulation promoted lung adenocarcinoma tumorigenesis via mTORC1 signaling pathway

Abstract

Background: Lung adenocarcinoma is a malignancy characterized by high mortality rates and unfavorable prognosis. However, the role of Leukotriene C4 Synthase (LTC4S) in lung cancer remains uninvestigated.

Methods: The expression and prognostic value of LTC4S in LUAD were analyzed using the GEPIA online database. Subsequently, the function of LTC4S in lung cancer cells was examined through gain-of function experiments, using assays to evaluate tumor malignant behavior. Subcutaneous xenograft experiments in vivo was used for investigating the functions of LTC4S. Then, tumor hallmark pathways were analyzed by GSEA. Western blot assay was used to validate the impact of LTC4S on mTORC1 pathway. Finally, the correlation of mRNA and methylation of LTC4S were analyzed by cBioPortal. qRT-PCR, ChIP-qPCR and ChIP-Atlas were used to verify the regulation factors of LTC4S low expression in LUAD cells.

Results: LTC4S presented significant decreased expression and favorable prognostic significance in LUAD. LTC4S was correlated with clinical stages in LUAD, which showed decreased expression gradually and significantly along with TNM stages. LTC4S-co-expressed genes were closely related to Ras signaling pathway, and MAPK signaling pathway. Overexpression of LTC4S inhibited cancer malignant phenotype and tumor growth in vitro and vivo. GSEA analysis and Western blot assay suggested low expression of LTC4S activated mTORC1 signaling pathway in LUAD. Moreover, the DNA methylation level of LTC4S in LUAD tissue was markedly elevated compared to normal tissue. The hypermethylation of the LTC4S promoter by DNMT3A leads to the decreased expression of LTC4S in LUAD.

Conclusions: In conclusion, low expression of LTC4S serves as an unfavorable prognostic marker and the critical function of LTC4S in controlling the progression of LUAD. This highlights the promise for exploring the clinical benefits of manipulating LTC4S in LUAD targeted therapies.

Keywords: DNA methylation; Leukotriene C4 Synthase (LTC4S); Lung adenocarcinoma (LUAD); Prognosis; mTORC1 signaling pathway.

Fig. 1

LTC4S showed significant decreased expression and favorable prognostic significance in lung adenocarcinoma. (A) The relative expression level of LTC4S in pan-cancer from TCGA database. (B) The overall survival (OS) and disease-free survival (DFS) of high/low expression LTC4S in TCGA database was analyzed by GEPIA. (C) Kaplan-Meier Plotter of high/low expression LTC4S based on GSE50081 and GSE30219 dataset.

Fig. 2

LTC4S was negatively correlated with clinical stages in lung adenocarcinoma. (A) The relative level of LTC4S in different stages (stages I, II, III, IV). (B) The relative level of LTC4S in different T stage (T1, T2, T3, T4). (C) The relative expression level of LTC4S in different N stage (N0, N1, N2-3). (D) The relative expression level of LTC4S in different M stage (M0, M1). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 3

Enrichment analysis of LTC4S co-expressed genes in LUAD. (A) Volcano map of co-expression genes associated with LTC4S. (B and C) Heat maps of the top 50 co-expression genes expression in LUAD, Pink represents LTC4S high expression, green represents LTC4S low expression. (D) GO terms enrichment analysis for LTC4S co-expression genes. (E) KEGG pathways of LTC4S co-expression genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Effect of LTC4S on cell proliferation, migration, and apoptosis of LUAD cells. (A) The relative expression level of LTC4S in normal cell line BEAS-2B, lung cancer cell line A549 and NCI–H1299. (B) The overexpression efficiency of LTC4S in A549 and NCI–H1299. (C) The proliferation capability of LTC4S overexpression A549 and NCI–H1299 cells was detected by CCK8 assay. (D) The apoptosis rate of LTC4S overexpression A549 and NCI–H1299 cells was detected by the flow cytometry assay. (E) Wound healing assay was conducted to explore the migration capability of LTC4S overexpression A549 and NCI–H1299 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5

Effect of LTC4S on tumor growth. (A) The tumor growth of Control and LTC4S OE groups was measured in vivo. (B) Tumor volume was measured at day7, day10, day13, day16, day19 and day21. (C) Tumor weight was measured at day21. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

Molecular mechanisms for the role of LTC4S in LUAD (A) The difference in the HALLMARKS pathway between high and low LTC4S group was analyzed by GSEA enrichment analysis. (B) The differences of KEGG pathway enrichment between high and low LTC4S group. (C) The hallmarks MYC targets and MTORC1 signaling pathways were enriched. (D) Western blotting assay was used to verify the MTORC signaling related protein (p-S6K1, p-S6, and p-4EBP1) expression in A549 and NCI–H1299 cells. (E) Relative expression of MTORC signaling related protein (p-S6K1, p-S6, and p-4EBP1) in A549 and NCI–H1299 cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7

Hyper-methylation contributes to reduced LTC4S expression. (A) The alteration frequency of LUAD is high according to LUAD datasets in CBioPortal. (B) The DNA methylation levels of LTC4S in LUAD compared to normal tissue. (C) The correlation of DNA methyltransferase 3A (DNMT3A) or DNMT3B and LTC4S. (D) The correlation of mRNA and methylation of LTC4S and tumor purity. (E) The LTC4S methylation level in different T stage (T1-T4). (F) A549 and NCI–H1299 cells were treated with 5-AZA-CdR for 48h, and LTC4S expression was examined by qRT-PCR assay. (G) LTC4S expression was analyzed by qRT-PCR after transfection of DNMT3A siRNA in A549 and NCI–H1299 cells. (H) The ChIP-qPCR assays showed that DNMT3A bond to the LTC4S promoter region. (I) The ChIP-Atlas analysis of enrichment of LTC4S promoter. *p < 0.05, **p < 0.01, ***p < 0.001.

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