The Overexpression of Acyl-CoA Medium-Chain Synthetase-3 (ACSM3) Suppresses the Ovarian Cancer Progression via the Inhibition of Integrin β1/AKT Signaling Pathway

Abstract

Ovarian cancer is considered as one of the most fatal gynecologic malignancies. This work aimed to explore the effects and regulatory mechanism of Acyl-CoA medium-chain synthetase-3 (ACSM3, a subunit of CoA ligases) in ovarian cancer progression. As well as employing CCK-8 assay, clone formation assay, and cell cycle analysis were carried out to investigate cell proliferation ability. Wound healing assay and transwell assay were subsequently used to assess cell migration and invasion. Mice xenografts were then conducted to measure the effects of ACSM3 on tumor development in vivo. Our bioinformatics analysis suggested that the expression of ACSM3 was down-regulated in ovarian cancer tissues, and the low expression level of ACSM3 might related with poorer overall survival than high mRNA expression of ACSM3 in ovarian cancer patients. We artificially regulated the expression of ACSM3 to evaluate its effects on ovarian cancer malignant phenotypes. Our data revealed that the overexpression of ACSM3 inhibited cell proliferation, migration, and invasion of ovarian cancer cells. In contrast, the knock-down of ACSM3 received the opposite results. Our western blot results showed that the Integrin β1/AKT signaling pathway was negatively regulated by ACSM3 expression. Moreover, ACSM3 overexpression-induced suppression of cell migration and invasion activities were abolished by the overexpression of ITG β1 (Integrin β1). Additionally, the growth of ovarian cancer xenograft tumors was also repressed by the overexpression of ACSM3. And ACSM3 interference obtained the contrary effects in vivo. In summary, ACSM3 acts as a tumor suppressor gene and may be a potential therapeutic target of ovarian cancer.

Keywords: acyl-CoA medium-chain synthetase-3; integrin β1; metastasis; ovarian cancer; proliferation.

Figure 1

The expression of ACSM3 and its co-relation of the overall survival of ovarian cancer. (A) Boxplot of ACSM3 in ovarian cancer. TPM, transcripts per kilobase of exon model per million mapped reads. (B) Overall survival time between patients with high and low ACSM3 expression. Dotted lines indicated the 95% confidence interval. HR, hazard ratio. (C) Relative protein expression of ACSM3 in ovarian epithelial cells and ovarian cancer cells: OV-90, SK-OV-3, OVCAR-3, and A2780 cells. (D) Relative protein expression of ACSM3 in Lv-shRNA-ACSM3 infected OV-90 cells (E) Relative protein expression of ACSM3 in Lv-ACSM3 infected A2780 or SK-OV-3 cells. Measurement data were expressed as mean ± SD of three independent experiments. ^^p < 0.01 versus ovarian epithelial cells; *p < 0.05, **p < 0.01 versus Lv-shRNA-NC; ^## p < 0.01 versus Lv-Vector.

Figure 2

Effects of ACSM3 on the proliferation of ovarian cancer cells. OV-90 cells were infected with ACSM3 shRNAs. A2780 or SK-OV-3 cells were infected with overexpressed ACSM3 vectors. (A–C) OV-90, A2780, or SK-OV-3 cells were subjected to CCK-8 assay. (D–F) The number of colonies. (G–I) Cell cycle arrest was detected by the flow cytometer. Measurement data were expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus Lv-shRNA-NC; ^#P < 0.05, ^##P < 0.01 versus Lv-Vector.

Figure 3

Effects of ACSM3 on migration and invasion of ovarian cancer cells. OV-90 cells were infected with Lv-shRNA-ACSM3. A2780 or SK-OV-3 cells were infected with Lv-ACSM3. (A-C) Cell migration ability was measured by wound healing assay. Scale bar represented 200 μm (D-F) The ability of cell invasion was detected by transwell assay. Scale bar represented 100 μm. Measurement data were expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus Lv-shRNA-NC; ^#P < 0.05, ^##P < 0.01 versus Lv-Vector.

Figure 4

Effects of ACSM3 on the Integrin β1/AKT signaling pathway in ovarian cancer. (A) The protein expression of Integrin β1, p-AKT, AKT, cyclin D1, c-Myc, Vimentin, and E-cadherin in OV-90 cells following ACSM3 knockdown. (B) The protein expression of Integrin β1, p-AKT, AKT, cyclin D1, c-Myc, Vimentin, and E-cadherin in A2780 cells following ACSM3 overexpression. Measurement data were expressed as mean ± SD of three independent experiments. **P < 0.01 versus Lv-shRNA-NC; ^##P < 0.01 versus Lv-Vector.

Figure 5

Effects of overexpressed ITG β1 (Integrin β1) on ACSM3 up-regulated ovarian cancer cells. (A) The protein expression of Integrin β1. (B) ACSM3 up-regulated A2780 cells were transfected by ITG β1 (Integrin β1) overexpression vector and were subjected to CCK-8 assay. (C, D) Relative wound width (Scale bar represented 200 μm). (E, F) Invasion ability. Scale bar represented 100 μm. (G) Relative protein expression of p-AKT, AKT, cyclin D1, c-Myc, Vimentin, and E-cadherin. Measurement data were expressed as mean ± SD of three independent experiments. ^$$p < 0.01 versus NC; ^&p < 0.05, ^&&p < 0.01 versus Lv-Vector+NC; ^@p < 0.05, ^@@p < 0.01 versus Lv-ACSM+NC.

Figure 6

Effects of ACSM3 on the growth of ovarian cancer tumor in vivo. A xenograft tumor model was built with OV-90 cells infected with Lv-shRNA1-ACSM3 or A2780 cells infected with Lv-ACSM3. (A, B) The volume of xenograft tumors. (C) The expression of ACSM3 and Integrin β1 was measured by immunohistochemistry. Scale bar represented 50 μm. (D, E) Relative protein expression of p-AKT, AKT, c-Myc, Vimentin, and E-cadherin. Measurement data were expressed as mean ± SD of six independent experiments. **p < 0.01 versus Lv-shRNA-NC; ^#p < 0.05, ^##p < 0.01 versus Lv-Vector.