ATP8B1 Knockdown Activated the Choline Metabolism Pathway and Induced High-Level Intracellular REDOX Homeostasis in Lung Squamous Cell Carcinoma

Abstract

The flippase ATPase class I type 8b member 1 (ATP8B1) is essential for maintaining the stability and polarity of the epithelial membrane and can translocate specific phospholipids from the outer membrane to the inner membrane of the cell. Although ATP8B1 plays important roles in cell functions, ATP8B1 has been poorly studied in tumors and its prognostic value in patients with lung squamous cell carcinoma (LUSC) remains unclear. By investigating the whole genomic expression profiles of LUSC samples from The Cancer Genome Atlas (TCGA) database and Tianjin Medical University Cancer Institute and Hospital (TJMUCH) cohort, we found that low expression of ATP8B1 was associated with poor prognosis of LUSC patients. The results from cellular experiments and a xenograft-bearing mice model indicated that ATP8B1 knockdown firstly induced mitochondrial dysfunction and promoted ROS production. Secondly, ATP8B1 knockdown promoted glutathione synthesis via upregulation of the CHKA-dependent choline metabolism pathway, therefore producing and maintaining high-level intracellular REDOX homeostasis to aggravate carcinogenesis and progression of LUSC. In summary, we proposed ATP8B1 as a novel predictive biomarker in LUSC and targeting ATP8B1-driven specific metabolic disorder might be a promising therapeutic strategy for LUSC.

Keywords: ATP8B1; CHKA; LUSC; REDOX.

Figure 1

Low expression of ATP8B1 promotes poor prognosis for the tumor by regulating metabolism. (A) The frequency of 13 LINE-1 insertions in LUSC tissue samples from the TJMUCH cohort. The occurrence frequency of L1-ATP8B1 was the highest in the group with poor prognosis (survival time ≤ 40). (B) We divided LUSC samples from the TJMUCH cohort into L1-ATP8B1⁺ and L1-ATP8B1⁻ groups according to the relative mRNA expression level of L1-ATP8B1. The transcriptome sequencing data indicated that ATP8B1 mRNA expression was downregulated in the L1-ATP8B1⁺ group. In 109 LUSC tissues, ATP8B1 expression was divided into high- and low-expression groups according to the median of the expression values, and a high ratio of L1-ATP8B1⁺ samples was observed in ATP8B1^low tumor tissues. (C) Low expression of ATP8B1 was significantly associated with poor prognosis (p = 0.0001). (D) Low expression of ATP8B1 was associated with poor prognosis in patients with LUSC at 5-year survival in the TCGA database (p = 0.041). (E) Pathway enrichment of differentially expressed genes was associated with ATP8B1 in 109 tissues. (F) Pathway enrichment of differentially expressed genes was associated with ATP8B1 in the TCGA database.

Figure 2

Interference of ATP8B1 promotes malignant progression in vitro and in vivo. (A) Expression of ATP8B1 in H520 and SK-MES-1 cells. All the RT-qPCR results shown for H520^SH-NC and H520^SH-ATP8B1 were relative mRNA expression values compared to wildtype H520 cell without virus infection for normalization while all the RT-qPCR results shown for SK-MES-1^SH-NC and SK-MES-1^SH-ATP8B1 were relative mRNA expression values compared to wildtype SK-MES-1 cells. (B) Cell proliferation results. (C) MTT assay results. (D) Cell apoptosis results. (E) Representative images of the wound healing assays. (F) Representative images of the trans-well invasion assays. (G) H520^SH-ATP8B1, SK-MES-1^SH-ATP8B1, and their control cells were subcutaneously injected into NOD-SCID mice as xenografts. Representative images of the forming tumors and the tumor volume growth curve at various time points are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; Scale bar: 100 μm.

Figure 3

Low expression of ATP8B1 activates choline kinase and promotes the production of phosphorylcholine. (A) Six pairs of H520^SH-ATP8B1 and H520^SH-NC samples were tested using metabolomics analysis. (B) KEGG pathway enrichment was carried out for different metabolites. (C) The cell metabolomics results showed that choline levels decreased and phosphorylcholine levels increased in the ATP8B1 knockdown cell lines. (D) Upregulated CHKA protein expression was detected in H520^SH-ATP8B1 at the protein level in both cell lines. Original blots see Figure S4. (E) The results showed the relative mRNA expression of CHKA from the transcriptome sequencing data of TJMUCH LUSC tissues. (F) IHC results in 46 cases of LUSC showed that CHKA was highly expressed in the ATP8B1^low group. (G) Multispectral fluorescent immunohistochemistry proved that in LUSC, CHKA was highly expressed in the ATP8B1^low group. * p < 0.05; ** p < 0.01. Scale bar: 50 μm; 100 μm.

Figure 4

Interfering with CHKA expression in H520^SH-ATP8B1 inhibited tumor progression. (A) Expression of CHKA in different cells at the mRNA level. All the RT-qPCR results shown for H520^SH-ATP8B1 and H520^{SH-ATP8B1-si-CHKAsi-CHKA} were relative mRNA expression values compared to H520^SH-NC for normalization. (B) Expression of CHKA in different cells at the protein level. (C) The ELISA results of phosphorylcholine and phosphatidylcholine. (D) Cell proliferation results. (E) Cell apoptosis results. (F) Representative images of the wound healing assays. (G) Representative images of the trans-well invasion assays. (H) Analysis of phosphorylated ERK and phosphorylated AKT by Western blot. Original blots see Figure S4. * p < 0.05; ** p < 0.01; *** p < 0.001; Scale bar: 100 μm.

Figure 5

CHKA maintains REDOX balance by regulating the level of intracellular GSH. (A) KEGG pathway enrichment analysis of differential genes in H520^SH-ATP8B1 and H520^SH-NC by transcriptome sequencing. (B) GO pathway enrichment analysis of differential genes in H520^SH-ATP8B1 and H520^SH-NC. (C) The level of intracellular GSH/GSSG. (D) Flow cytometry results of intracellular ROS. (E) Cell proliferation results. (F) Cell apoptosis results. (G) Representative images of the wound healing assays. (H) Representative images of the trans-well invasion assays. (I) Analysis of phosphorylated ERK and AKT by Western blot. Original blots see Figure S4. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Scale bar: 100 μm.