Downregulation of GLYAT Facilitates Tumor Growth and Metastasis and Poor Clinical Outcomes Through the PI3K/AKT/Snail Pathway in Human Breast Cancer

Abstract

Background: The Glycine N-acyltransferase (GLYAT) gene encodes a protein that catalyzes the transfer of acyl groups from acyl CoA to glycine, resulting in acyl glycine and coenzyme A. Aberrant GLYAT expression is associated with several malignant tumors, but its clinical importance in human breast cancer (BC), has yet to be fully addressed. This study aims to evaluate the clinical function of GLYAT in BC patients.

Methods: GLYAT expression was determined by immune blot and immunohistochemistry in three BC cell lines and primary cancer tissues. The MDA-MB 231 cell line was used for GLYAT gene knockdown experiments while the MCF7 cell line for overexpression experiments. Colony formation experiments, soft agar experiments, and transwell assays were utilized for further inspection of cell proliferation and migration capabilities. Immunofluorescence and western blot were used to detect markers of the epithelial-mesenchymal transition (EMT) and changes in the PI3K/AKT/Snail pathway. The role of GLYAT in tumor growth and metastasis was also assessed in nude mice in vivo. Also, a correlation analysis was performed between clinicopathological features and GLYAT expression in BC patients.

Results: GLYAT was decreased in human BC tissues and cell lines. Functional analysis showed that knockdown of GLYAT augmented BC cell proliferation in vitro and in vivo. However, this phenomenon was reversed when GLYAT was overexpressed in the transfected cells. Moreover, downregulation of GLYAT promoted the migratory properties of BC cells, likely through the activation of PI3K/AKT/Snail signaling, which subsequently induced the EMT. IHC analysis indicated that GLYAT was decreased in human BC tissues and lower GLYAT expression was correlated with histological grade, tumor TNM stage, Ki-67 status, and poorer survival in BC patients. Furthermore, lower GLYAT expression seemed as an independent risk factor related to poor prognosis in BC patients based on Cox regression analyses.

Conclusion: Our findings demonstrate that downregulation of GLYAT expression in human breast cancer is correlated with EMT via the PI3K/AKT/Snail pathway and is also associated with histological grade, tumor TNM stage, Ki-67 status, and poor survival in breast cancer patients.

Keywords: EMT; GLYAT; PI3K/AKT; breast cancer; clinicopathological features; prognosis.

Figure 1

Characterization of GLYAT expression profiling in human breast cancer. (A) Heatmaps showing the clustering patterns of differentially expressed GLYAT between normal and breast cancer specimens. (B) Box plot analysis of the GLYAT mRNA levels in IDBC, ILBC and normal breast tissue samples. (C) Box plot analysis of the GLYAT mRNA levels in BC and NC tissues. T, tumor; N, normal. (D, E) Kaplan-Meier plots of OS in BC patients with different levels of GLYAT. *p < 0.05.

Figure 2

GLYAT suppresses BC cell proliferation and metastasis. (A) GLYAT protein level was assessed in different BC cell lines. (B) MDA-MB-231 cells were transfected with GLYAT KD plasmid and a scrambled plasmid as control. The suppression of GLYAT in MDA-MB-231 cells was confirmed at the protein level. (C) MCF-7 cells were transfected with GLYAT OE plasmid and a scrambled plasmid as control. The over expression of GLYAT in MCF-7 cells was confirmed at the protein level. (D, E) GLYAT KD in MDA-MB-231 cells significantly increased colony numbers and size compared with the scramble cells. (F) Transwell assay revealed that the migration ability of MDA-MB-231 cells was significantly increased following transfection with GLYAT KD1 and KD2 compared with the scramble control. (G) Transwell assay revealed that the migratory ability was significantly decreased in MCF-7 cells GLYAT OE compared with scramble control. **p < 0.01. Scale bars for (E) are 100μm and 25μm, and for (F, G) are 100μm.

Figure 3

GLYAT suppress EMT phenotype in BC cells. (A, B) Immunofluorescence assay showed that E-cadherin expression was decreased and the vimentin expression was increased after the treatment of GLYAT KD. (C–G) Protein levels of E-cadherin was reduced, whereas the levels of Vimentin, N-cadherin, and Fibronectin were increased in MDA-MB-231 cells following GLYAT inhibition. (H–J) Protein levels of E-cadherin was increased whereas the expression of Vimentin was reduced in GLYAT OE MCF-7 cells. *p < 0.05, **p < 0.01. Scale bars are 25μm.

Figure 4

GLYAT regulated breast cancer cells EMT progression through PI3K/Akt signaling Pathway. (A–E) Protein levels of p-AKT,AKT, PI3K and SNAI1 were significantly activated in GLYAT KD MDA-MB-231 cells. (F–J) Protein levels of p-AKT,AKT, p-PI3K and SNAI1 were significantly reduced in GLYAT over expressed OE MF-7 cells. *p < 0.05, **p < 0.01, and p < 0.05 was considered to be statistically significant.

Figure 5

GLYAT suppress breast cancer proliferation, metastasis and EMT in vivo. (A) Schematic diagram of the metastasis model in mice. (B–D) The mice injected with stable GLYAT KD MDA-MB-231 cells had markedly bigger and heavier tumors. (E–G) The mice injected with stable GLYAT OE MCF-7 cells had markedly smaller and lighter tumors. (H) Immunofluorescence assay of serial sections mouse tumor tissues revealed that GLYAT KD MDA-MB-231 cells significantly decreased the expression of E-cadherin whereas increased the expression of Vimentin and p-AKT. (I) Immunofluorescence assay of serial sections mouse tumor tissues revealed that, in GLYAT OE MCF-7 cells, the expression of E-cadherin was increased whereas Vimentin and p-AKT were decreased. (J) The schematic diagram of the role of GLYAT in BC. *p < 0.05, **p < 0.01. Scale bars are 50μm.

Figure 6

GLYAT is less expressed in BC tissues and correlated with worse survival. (A, B) Immunohistochemical staining showed that GLYAT was downregulated in BC tissues compared with NC. (C) Immunohistochemical staining showed that the lower differentiation of the breast cancer, the less expressed of GLYAT in breast cancer tissues. (D) Kaplan-Meier analysis of DFS in BC patients with different levels of GLYAT. (E–H) Kaplan-Meier analysis of DFS in luminal A, luminal B, HER2 positive and triple negative BC patients with different levels of GLYAT. (I–L) Kaplan-Meier analysis for DFS by Ki67, ER, PR, HER2 status in BC patients with different levels of GLYAT. *p < 0.05, **p < 0.01. Scale bars for (A) are 200μm, and for (B) are 50μm and 200μm.