Inhibition of FASN suppresses the malignant biological behavior of non-small cell lung cancer cells via deregulating glucose metabolism and AKT/ERK pathway

Abstract

Background: Fatty acid synthase (FASN) is overexpressed in most human carcinomas, including non-small cell lung cancer (NSCLC), and contributes to poor prognosis. An increasing number of studies have highlighted the potential function of FASN as both a biomarker and therapeutic target for cancers. However, the underlying molecular mechanisms of FASN in glucose metabolism and the malignant biological behavior of NSCLC remain the subjects of intensive investigation.

Methods: FASN expression was depleted by FASN-siRNA in A549 and NCI-H1299 cell lines to detect the function of glucose metabolism and the malignant biological behavior of NSCLC cells. Western-blot and qPCR were applied to determine the expressions of FASN, t-AKT, p-AKT, t-ERK, p-ERK, PKM2, HK2 and AZGP1. ATP and lactate were detected to determine the activation of glucose metabolism. CCK8 and transwell assays were used to detect the proliferation, invasion, and migration capacity of the two types of NSCLC cells. The xenograft mouse model was used to evaluate tumor weights after suppression of FASN.

Results: LV-FASN-siRNA and its control lentiviral vector were successfully transfected into the two types of NSCLC cells (A549 and NCI-H1299). LV-FASN siRNA significantly suppressed FASN expression in both NSCLC cell types, and expressions of p-AKT, p-ERK, PKM2, and AZGP1 were also significantly decreased. Notably, the levels of ATP and lactate were significantly decreased after transfection with LV-FASN siRNA. The proliferation of both NSCLC cell types was decreased after suppression of FASN. The invasion and migration capacity of A549, but not NCI-H1299, were inhibited following down-regulation of FASN. In vivo, inhibition of FASN caused a marked animal tumor weight loss.

Conclusions: FASN was involved in glucose metabolism via down-regulation of the AKT/ERK pathway and eventually altered the malignant phenotype in lung cancer cells.

Keywords: AKT/ERK pathway; Fatty acid synthase; Glucose metabolism; NSCLC; Xenograft.

Fig. 1

Target genes’ relative expressions detected by RT-qPCR after LV–siFASN transfection. The relative expressions of target genes in each group are shown relative to those of the blank group. FASN mRNA expressions were significantly decreased in the experimental group of A549 cells (P < 0.05; a) and NCI-H1299 cells (P < 0.05; b), suggesting that LV–FASN RNAi successfully suppressed the expression of FASN. Additionally, AZGP1 expression was lower under FASN deficiency in A549 cells (P < 0.05; a) and NCI-H1299 cells (P < 0.05; b). The differences between the Akt-mRNA and ERK-mRNA expressions of each group of A549 cells (a) and NCI-H1299 cells (b) were not statistically significant. * compared to the control group (P < 0.05). # compared to the blank group (P < 0.05)

Fig. 2

Inhibition of FASN expression suppresses the activity of the Akt/ERK signaling pathway. Target protein expression of A549 cells (left panel) and NCI-H1299 cells (right panel) were detected by western-blot. Compared to the blank and control groups, the expressions of p-Akt and p-ERK in the experimental group were significantly inhibited. However, there were no statistically significant differences in the t-Akt and t-ERK expressions of each group. Importantly, the expression of PKM2 but not HK2 (both are key enzymes in glucose metabolism) was dramatically lower in the experimental group. * compared to the control group (P < 0.05). # compared to the blank group (P < 0.05)

Fig. 3

Inhibition of FASN suppresses the activity of glucose metabolism. The levels of ATP (a-b) and lactic acid (c-d) were detected by the corresponding kits. ATP levels in the experimental group were significantly lower than those of the blank and control groups (P < 0.05). The concentration of lactic acid in the experimental group was also lower in the two types of NSCLC cell lines

Fig. 4

Inhibition of FASN suppresses the proliferation of NSCLC cells. Cell viability assays were conducted 1–5 days after RNAi transfection. Compared with the blank and control groups, the proliferation of experimental group A549 cells was significantly lower at the fifth day post-transfection (a); and experimental group NCI-H1299 cells were significantly lower 4 days post-transfection (b). * P < 0.05, ** P < 0.01: compared to the control group. # P < 0.05, ## P < 0.01: compared to the blank group

Fig. 5

Inhibition of FASN suppresses the migration and invasion ability of A549 cells. A549 (a) and NCI-H1299 (b) cells were seeded into the upper chamber of a transwell system. The cells in the lower chamber were stained and counted to assess the migration and invasion ability of the target cells. Compared with the blank and control groups, the numbers of A549 cells were significantly lower (c); however, there were no statistically significant differences between the numbers of NCI-1299 cells in the experimental group and the other groups (d)

Fig. 6

Inhibition of FASN blocks xenograft tumor growth of NSCLC in nu/nu mice. A preliminary experiment was conducted to detect the tumorigenicity ability of wild-type A549 cells (a). Three groups of A549 cells were subcutaneously injected into nu/nu mice and xenograft tumors were harvested after 3 weeks (b), The tumor weight of the experimental group was significantly lower than those of the blank and control groups (c). The scale bars of (a) and (b) is 1 cm