LPCAT1 reprogramming cholesterol metabolism promotes the progression of esophageal squamous cell carcinoma

Abstract

Tumor cells require high levels of cholesterol for membrane biogenesis for rapid proliferation during development. Beyond the acquired cholesterol from low-density lipoprotein (LDL) taken up from circulation, tumor cells can also biosynthesize cholesterol. The molecular mechanism underlying cholesterol anabolism in esophageal squamous cell carcinoma (ESCC) and its effect on patient prognosis are unclear. Dysregulation of lipid metabolism is common in cancer. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been implicated in various cancer types; however, its role in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we identified that LPCAT1 is highly expressed in ESCC and that LPCAT1 reprograms cholesterol metabolism in ESCC. LPCAT1 expression was negatively correlated with patient prognosis. Cholesterol synthesis in ESCC cells was significantly inhibited following LPCAT1 knockdown; cell proliferation, invasion, and migration were significantly reduced, along with the growth of xenograft subcutaneous tumors. LPCAT1 could regulate the expression of the cholesterol synthesis enzyme, SQLE, by promoting the activation of PI3K, thereby regulating the entry of SP1/SREBPF2 into the nucleus. LPCAT1 also activates EGFR leading to the downregulation of INSIG-1 expression, facilitating the entry of SREBP-1 into the nucleus to promote cholesterol synthesis. Taken together, LPCAT1 reprograms tumor cell cholesterol metabolism in ESCC and can be used as a potential treatment target against ESCC.

Fig. 1. LPCAT1 is highly upregulated in esophageal squamous cell carcinoma and correlates with poor patient prognosis.

A Volcano plot compared the expression fold changes of the genes for ESCC tissues versus adjacent normal tissues. The red dots represent the genes with significantly changed expression level. B Clustered heat map for all genes with altered expression, with rows representing gene and columns representing tissues. C, D Expression levels of LPCAT1 in ESCC tumor and normal tissues were determined using western blotting analysis. Data are shown as relative expression means and P value from three independent experiments. E Expression levels of LPCAT1 in tumors were determined using immunohistochemistry analysis. Histograms show the means and P value from three independent experiments. F Statistical analyses of the LPCAT1 in tumor tissues. G The mRNA of LPCAT1 in 185 ESCC and normal tissues was determined using qRT-PCR, **P < 0.01 via unpaired t test. H The protein of LPCAT1 in 185 ESCC and normal tissues was determined using immunohistochemistry analysis, **P < 0.01 via unpaired t test. I Statistical analyses of the association between LPCAT1 expression and survival in patients with ESCC. J Statistical analyses of the association of LPCAT1 expression with survival probability in ESCC patients. K Serum LPCAT1 levels in 71 healthy subjects and 154 patients with ESCC. P < 0.01 via unpaired t test. L ROC curve analysis of the value of LPCAT1 in ESCC diagnosis. *P < 0.05. **P < 0.01.

Fig. 2. LPCAT1 promotes ESCC cell proliferation, migration, and invasion.

A–D PCR and western blotting showing the expression of LPCAT1 in EC9706 cells (A, B) and TE1 cells (C, D) transfected with si-NC and si-LPCAT1. Data are shown as relative expression means and P value from three independent experiments. E, F The proliferative ability of EC9706 cells (E) and TE1 cells (F) after transfection was evaluated using CCK-8 assay. G, H Colony-formation assay for LPCAT1-knockdown EC9706 cells and TE1 cells (G) and quantitative analysis of LPCAT1 in each group (H). I, J Transwell chamber assays for LPCAT1-depletion ESCC cells. The average numbers of migrated cells were counted after 24 h incubation and expressed as mean ± S.D. K, L Matrigel invasion assays for LPCAT1-depletion ESCC cells. The average numbers of migrated cells were counted after 24 h incubation and expressed as mean ± S.D. Data are from three independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA).

Fig. 3. LPCAT1 inhibits apoptosis of ESCC cells and promotes cell cycle progression and anoikis resistance.

A, B Annexin V-FITC/PI double staining was used to assess the apoptosis of EC9706 cells and TE1 cells after transfection with si-NC and si-LPCAT1. C, D DNA content of EC9706 cells and TE1 was analyzed by flow cytometry after transfection with si-NC and si-LPCAT1. E, F EC9706 cells and TE1 cells were stably transfected with LPCAT1 lentivirus. G Confocal assay was performed to assess the expression of LPCAT1 in EC9706 cells and TE1 cells. H, I Colony-formation assay was performed to assess EC9706 cells and TE1 cells for anoikis resistance. The average size of colonies was calculated as mean ± S.D. **P < 0.01 (unpaired t test). Data are from three independent experiments.

Fig. 4. LPCAT1 promote the expression of cholesterol synthesis signal pathway genes in esophageal squamous cell carcinoma.

A Volcano plots showing that, among differentially expressed genes, 23 were upregulated and 27 were downregulated in EC9706 cells after knockdown of LPCAT1. B A cluster heat map was used to show the expression variations of these gene transcripts in EC9706 cells. C Volcano plots illustrating that, among differentially expressed genes, 9 were upregulated and 17 were downregulated in TE1 cells after knockdown of LPCAT1. D A cluster heat map was used to show the expression variations of these gene transcripts in TE1 cells. E Venn analysis of genes with altered expression in EC9706 and TE1 cells after knockdown of LPCAT1. F, G All of the genes altered in EC9706 and TE1 cells were subject to GO analysis. H All genes with altered expression were enriched and analyzed by human gene signaling pathway.

Fig. 5. LPCAT1 promote ESCC cholesterol synthesis by EGFR/INSIG-1/SREBP-1 pathway.

A The level of cholesterol in EC9706 and TE1 cells transfected with sh-control and sh-LPCAT1 was detected using the cholesterol detection kit. B The expression of SQLE, Insig-1, and MSMO1 in EC9706 and TE1 cells transfected with sh-control and sh-LPCAT1 was detected using western blot. C, D The expression of p-SREBP-1 in the nucleus in EC9706 and TE1 cells transfected with sh-control and sh-LPCAT1 was detected using western blot and immunofluorescence. E The expression of p-EGFR and p-PI3K in EC9706 and TE1 cells transfected with sh-control and sh-LPCAT1 was detected using western blot. F The expression of SQLE, Insig-1, and MSMO1 in EC9706 and TE1 cells treated with NSC228155 or 740Y-P after transfection with sh-LPCAT1 was detected using western blot. G The expression of p-SREBP-1 in the nucleus in EC9706 and TE1 cells treated with NSC228155 or 740Y-P after transfection with or without sh-LPCAT1 was detected using western blot. H The level of cholesterol in EC9706 and TE1 cells treated with NSC228155 or 740Y-P after transfection with or without sh-LPCAT1 was detected using the cholesterol detection kit. Data represent three independent experiments. *P < 0.05, **P < 0.01 (unpaired t test, one-way ANOVA). Data are from three independent experiments.

Fig. 6. PI3K signaling promotes cholesterol synthesis by upregulation of SQLE transcriptional activity via SP1 and SREBF2.

A, B To explore the key transcriptional activity region, serial truncated plasmids of the SQLE promoter and the pRL-TK plasmid were transfected into TE1 cells for a dual-luciferase reporter assay. C DMSO and 20 μM 740Y-P were added after SQLE-truncated plasmids and the control plasmid were co-transfected and the promoter activity was measured with the dual-luciferase reporter assay. D The transcription factor-binding sites on the 5‘-regulatory region sequence of SQLE were predicted using the JASPAR online software. E The mutant plasmids were transiently transfected into HeLa cells and the luciferase activity was measured after 48 h. F The colorful letters indicate the motif of transcription factor-binding site of SP1 and SREBF2 cited from JASPAR. G The transcriptional activity of SQLE was elevated after overexpression of SP1 and SREBF2. H Chip-PCR detection of SP1 and SREBF2 binding in the promoter of SQLE. I, J The expression levels of SP1 and SREBF2 in nuclear was detected using western blot (I) and immunofluorescence (J) after knocking down of LPCAT1. K The expression of SREBF2 and SP1 in EC9706 and TE1 cells treated with NSC228155 or 740Y-P after transfection with or without sh-LPCAT1 was detected using western blot. L TE1 cells treated with the PI3K agonist (20 μM) or DMSO were subjected to a ChIP assay. IgG was served as a negative control. The results from three independent experiments. M The expression of SQLE and nucleus SP1 and SREBF2 after overexpression of SP1 and SREBF2 in TE1 cells that were transfected with or without sh-LPCAT1 was detected using western blot. Normalized luciferase activity and P values are from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test, one-way ANOVA). Data are from three independent experiments.

Fig. 7. LPCAT1 promote ESCC development and cholesterol synthesis in vivo.

EC9706 and TE1 cells stably transfected with sh-control and sh-LPCAT1 were injected subcutaneously into NOD/SCID mice. Four weeks after the injection, mice were sacrificed by carbon dioxide suffocation. A Representative bioluminescent/photographic images in the nude mouse. B Quantitation of signals plotted against time points. C Representative images of tumor nodes were shown. D, E Tumor growth (D) and survival curves (E) are plotted. F Cholesterol was detected using the cholesterol detection kit in the tumor tissues of each group. Data are from three independent experiments. *P < 0.05, **P < 0.01 (unpaired t test). Data are from three independent experiments.