FFAR4 activation inhibits lung adenocarcinoma via blocking respiratory chain complex assembly associated mitochondrial metabolism

Abstract

Despite notable advancements in the investigation and management of lung adenocarcinoma (LUAD), the mortality rate for individuals afflicted with LUAD remains elevated, and attaining an accurate prognosis is challenging. LUAD exhibits intricate genetic and environmental components, and it is plausible that free fatty acid receptors (FFARs) may bridge the genetic and dietary aspects. The objective of this study is to ascertain whether a correlation exists between FFAR4, which functions as the primary receptor for dietary fatty acids, and various characteristics of LUAD, while also delving into the potential underlying mechanism. The findings of this study indicate a decrease in FFAR4 expression in LUAD, with a positive correlation (P < 0.01) between FFAR4 levels and overall patient survival (OS). Receiver operating characteristic (ROC) curve analysis demonstrated a significant diagnostic value [area under the curve (AUC) of 0.933] associated with FFAR4 expression. Functional investigations revealed that the FFAR4-specific agonist (TUG891) effectively suppressed cell proliferation and induced cell cycle arrest. Furthermore, FFAR4 activation resulted in significant metabolic shifts, including a decrease in oxygen consumption rate (OCR) and an increase in extracellular acidification rate (ECAR) in A549 cells. In detail, the activation of FFAR4 has been observed to impact the assembly process of the mitochondrial respiratory chain complex and the malate-aspartate shuttle process, resulting in a decrease in the transition of NAD⁺ to NADH and the inhibition of LUAD. These discoveries reveal a previously unrecognized function of FFAR4 in the negative regulation of mitochondrial metabolism and the inhibition of LUAD, indicating its potential as a promising therapeutic target for the treatment and diagnosis of LUAD.

Keywords: FFAR4; LUAD; Metabolism reprogramming; OXPHOS.

Fig. 1

FFAR4 expression is significantly decreased in LUAD. A FFAR4 levels in different tumors were assessed using TCGA database. B The median expression of FFAR4 between normal and tumor samples in bodymap from the GEPIA. C Tumor tissue and control tissue samples in TCGA database were compared for FFAR4 expression. D A comparison of FFAR4 expression between normal tissue samples from GTEx and control and normal tissue samples from TCGA database was performed using WRST. E–F Analysis of GEO-GSE40275 and GEO-GSE118370 datasets confirms the presence of FFAR4 mRNA deregulation in LUAD. G–H Expression of FFAR4 mRNA in lung adenocarcinoma and paracarcinoma tissues of 11 patients with LUAD from the First Affiliated Hospital of Ningbo University. Data are expressed as the mean ± SEM. P < 0.05 was considered statistically significant using two-way analysis of variance (ANOVA); NS, not significant

Fig. 2

Relationship between FFAR4 expression and patient overall survival in patients with LUAD. A Kaplan–Meier plot of the expression level of FFAR4 in the overall cohort of patients with LUAD. B–I Subgroup analyses for patients with T stage: T3 and T4; N stage: N0, N1, and N2; M stage: M0; pathological stage: stage I, II, and III; pathological stage: stage II; residual tumor: R0 and R1; anatomic neoplasm subdivision: left; and age: ≤ 65 years old. J A nomogram that predicts the 1-, 3-, and 5-year survival rate of patients with LUAD

Fig. 3

ROC analysis suggests FFAR4 is valuable molecular biomarker for LUAD. A ROC curves indicated that FFAR4 expression was a valuable indicator of the difference between tumors and normal tissues. The X- and Y-axis correspond to rates of true- and false-positive results, respectively. B–E Subgroup analyses for stage I, II, III, and IV LUAD tumors

Fig. 4

FFAR4 activation inhibits lung adenocarcinoma cell proliferation and induces metabolic shift in A549 cells. A Comparison of FFAR4 expression in three cell lines. B The effect of different concentration of TUG891 on proliferation of A549 cells. C Effects of 40 μM TUG891 treatment on A549 cell proliferation at 24, 48, and 72 h. D Effects of FFAR4 activation on cell cycle transition in A549 cells. E Color changes in A549 medium treated with different concentrations of TUG891. F Changes in the lactate content of the medium after treatment with 40 μM TUG891. G–J Effect of 40 μM TUG891 agonist on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in A549 cells. Data are expressed as the mean ± SEM. P < 0.05 was considered statistically significant using two-way ANOVA; NS, not significant

Fig. 5

FFAR4 activation leads to reduction of NAD⁺/NADH ratio by affecting mitochondrial complex I assembly. A–C Tree plot, dot plot, and cnet plot of the results of ORA (GO analysis) of downregulated genes after FFAR4 activation. The size of the dots indicates the number of genes in a particular pathway and the color indicates the correlation with the adjusted P value. D–F NADH and NAD⁺ levels and the NAD⁺/NADH ratio were measured from A549 cells between the vehicle group and FFAR4 activator group

Fig. 6

FFAR4 activation enhances malate–aspartate shuttling in A549 cells. A Overall differences between groups were demonstrated by the using orthogonal partial least-squares discriminant analysis (OPLS-DA). B A random forest approach was used to select the features with the highest contribution to the accuracy of the sample grouping predictions. C Based on the significantly different metabolites, ORA was used to find KEGG pathways that were significantly enriched in metabolites. D Conceptual summary