From mitochondria to tumor suppression: ACAT1's crucial role in gastric cancer

Abstract

Acetyl CoA acetyltransferase 1 (ACAT1), a mitochondrial enzyme, is mainly involved in the formation and decomposition of ketones, isoleucine, and fatty acids. Previous clinical studies showed that mutations in the ACAT1 gene lead to ketoacidosis, Notably the role of ACAT1 in human cancer' pathogenesis varies depending on cancer type, and its specific role in gastric cancer remains largely unknown. In the current study, we found that the expression of ACAT1 in primary late-stage gastric cancer tumor tissues was significantly lower than in early-stage tumors. This observation was further confirmed in high-grade gastric cancer cell line MKN45. The expression of CD44 and OCT4 was decreased, while CD24 expression was increased by overexpressing ACAT1 in MKN45 gastric cancer cells. Moreover, the ability of gastric cancer cells to form colonies on soft agar was also reduced by ACAT1 overexpression. Likewise, overexpression of ACAT1 inhibited epithelial mesenchymal transition (EMT) in gastric cancer cells evidenced by increased expression of the epithelial marker E-Cadherin, decreased expression of mesenchymal marker vimentin, and decreased expression levels of SNAI 1/3. In addition, ACAT1 overexpression inhibited cell migration and invasion, improved the response to 5-Fluorouracil (5-FU) and etoposide. In contrast, inhibition of ACAT1 activity promoted the proliferation of gastric cancer cells. The xenotransplantation results in nude mice showed that overexpression of ACAT1 in gastric cancer cells inhibited tumor growth in vivo. In addition, the low expression of ACAT1 in gastric cancer was further validated by searching public databases and conducting bioinformatic analyses. Mechanistically, bioinformatic analysis found that the inhibitory effect of ACAT1 in gastric cancer may be related to the Adipocytokine Signaling Pathway, Ppar Signaling Pathway, Propanoate Metabolism and P53 Signaling Pathway. Correlation analysis indicated ACAT1 mRNA expression was correlated with immune infiltrates. Collectively, our data show that ACAT1 induces pronounced inhibitory effects on gastric cancer initiation and development, which may impact future strategies to treat this aggressive cancer.

Keywords: ACAT1; EMT; gastric cancer; mitochondrial enzyme; tumor stem cells.

Figure 1

The relative expression of ACAT1 (cancer tissue/paracancerous tissue) was significantly decreased in patients with advanced gastric cancer. (A) Western blot analysis of protein expression of ACAT1 in gastric cancer patient tumor tissues at different stages. A: gastric cancer tissue, B: adjacent noncancerous area. (B) The proportion of patients with high expression of ACAT1 and those with low expression of ACAT1 in 88 pairs of samples with stage I-IV gastric cancer. (C, D) Relative expression of 88 pairs of gastric cancer patient samples. Relative expression = the normalized value of cancer tissue/the normalized value of adjacent tissue. Values are means ± SD. *p < 0.05, ***p < 0.001.

Figure 2

The expression of ACAT1 in gastric cancer cell lines MKN45, N87 and AGS. (A) RT-qPCR analysis of ACAT1 mRNA. (B, C) Western blot analysis of ACAT1. (D) Immunofluorescence staining of ACAT1 (green). The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n = 3 in each group. Values are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

ACAT1 modulates the expression of stem cell markers. (A, B) RT-qPCR analysis of the mRNA expression of CD44 and OCT4 in gastric cancer cell lines MKN45, N87 and AGS. (C–E) RT-qPCR and Western blot to verify the overexpression of ACAT1 in MKN45 cells. (F, G) RT-qPCR analysis of CD44 and OCT4 in MKN45-ACAT1 cells and MKN45-LV5 cells. (H) Flow cytometric analysis of CD44 and CD24 in MKN45-ACAT1 and MKN45-LV5 cells. (I) Immunofluorescence staining of CD44 (green) and CD24 (red) in MKN45-ACAT1 and MKN45-LV5 cells. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n = 3 in each group. Values are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

ACAT1 impedes the EMT process by inhibiting the expression of SNAI1 and SNAI3. (A) Immunofluorescence staining of the EMT markers E-cadherin (green) and vimentin (red) in gastric cancer cell lines MKN45, N87 and AGS. The nuclei were stained with DAPI (blue). Scale bar: 200 μm. (B–D) Western blot was performed to analyze the levels of E-Cadherin and vimentin expression in MKN45-ACAT1 cells compared to MKN45-LV5 cells. (E) Immunofluorescence staining of E-Cadherin (green) and vimentin (red) in MKN45-ACAT1 and MKN45-LV5 cells. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. (F) RT-qPCR analysis of EMT-related genes in MKN45-ACAT1 and MKN45-LV5 cells. n = 3 in each group. Values are mean ± SD. ns, not significant. *p < 0.05, ***p < 0.001.

Figure 5

ACAT1 inhibits the migration and invasion of gastric cancer cells. (A–D) Transwell assay to assess the migration and invasion ability of MKN45-ACAT1 and MKN45-LV5 cells. Scale bar: 100 μm. n = 3 in each group. Values are mean ± SD. ***p < 0.001.

Figure 6

ACAT1 overexpression improves the response to chemotherapy drugs and inhibits the ability of gastric cancer cells to form colonies in soft agar. (A, B) The sensitivity of MKN45-ACAT1 and MKN45-LV5 cells to the chemotherapeutic drugs 5-FU (4 mM) and etoposide (80 μM) was analyzed by CCK8 reagent. (C) The sensitivity of AGS cells to the arecoline hydrobromide (200 μM) was analyzed by CCK8 reagent. (D) The anchorage-independent proliferation of AGS and control MKN45 cells was assessed through a soft agar colony formation assay. The scale bar in the 0.5X represents 5 mm, the scale bar in the 1.25X represents 2 mm and the scale bar in the 11.5X represents 200 μm. (E) The number of colonies formed by AGS and MKN45 cells. (F) The anchorage-independent growth of MKN45-ACAT1 and MKN45-LV5 cells was detected by using a soft agar colony formation assay. The scale bar in the 0.5X represents 5 mm, the scale bar in the 1.25X represents 2 mm and the scale bar in the 11.5X represents 200 μm. (G) The number of colonies formed by MKN45-ACAT1 and MKN45-LV5 cells. n = 3 in each group. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

ACAT1 overexpression inhibits tumor growth in nude mouse xenograft model. (A) MKN45-LV5 cells or MKN45-ACAT1 cells were injected into each nude mouse, and representative images were taken on day 21 after injection. Upper panel represents MKN45-LV5; lower panel represents MKN45-ACAT1. (B) The tumor volumes were measured on day 6, 9, 12, 15, 17, 19 and 21. (C, D) On day 21, the tumors were taken out, excised, and weighed. Upper panel represents MKN45-LV5; lower panel represents MKN45-ACAT1. n = 6 in each group. Values are mean ± SD. *p < 0.05, **p < 0.01.

Figure 8

Expression of the ACAT1 gene to the expression pattern of whole genes and bioinformatics analysis. (A) A volcano map based on ACAT1 expression patterns illustrating the differentially expressed genes (DEGs). (B) The expression level of the ACAT1 gene was used to generate a heat map that displays 20 genes that were either upmodulated or downmodulated. (C–F) The findings of the gene set enrichment analysis (GSEA). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9

Relationship between the ACAT1 gene expression and immune cell infiltration. (A) The relationship between the ACAT1 gene expression and immune cell infiltration status. (B–D) Differences in the degree to which certain immune cell subsets were enriched in the ACAT1 gene high- and low-expression groups. (E–J) Relationships between the ACAT1 gene expression and tumor microenvironment characteristics. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.