Long-Chain Acyl Coenzyme A Dehydrogenase, a Key Player in Metabolic Rewiring/Invasiveness in Experimental Tumors and Human Mesothelioma Cell Lines

Abstract

Cross-species investigations of cancer invasiveness are a new approach that has already identified new biomarkers which are potentially useful for improving tumor diagnosis and prognosis in clinical medicine and veterinary science. In this study, we combined proteomic analysis of four experimental rat malignant mesothelioma (MM) tumors with analysis of ten patient-derived cell lines to identify common features associated with mitochondrial proteome rewiring. A comparison of significant abundance changes between invasive and non-invasive rat tumors gave a list of 433 proteins, including 26 proteins reported to be exclusively located in mitochondria. Next, we analyzed the differential expression of genes encoding the mitochondrial proteins of interest in five primary epithelioid and five primary sarcomatoid human MM cell lines; the most impressive increase was observed in the expression of the long-chain acyl coenzyme A dehydrogenase (ACADL). To evaluate the role of this enzyme in migration/invasiveness, two epithelioid and two sarcomatoid human MM cell lines derived from patients with the highest and lowest overall survival were studied. Interestingly, sarcomatoid vs. epithelioid cell lines were characterized by higher migration and fatty oxidation rates, in agreement with ACADL findings. These results suggest that evaluating mitochondrial proteins in MM specimens might identify tumors with higher invasiveness. Data are available via ProteomeXchange with the dataset identifier PXD042942.

Keywords: biomarker; fatty acid β-oxidation; long-chain specific acyl-CoA dehydrogenase; malignant mesothelioma; metabolism; mitochondria.

Figure 1

Histological features of the four experimental rat mesothelioma tumor models. HPS staining, ×400 (the scale bar represents 50 µm). Inserts (bottom right corner) represent general views (the scale bars represent 5 mm (left column) or 2.5 mm (right column)), with the open red arrows showing the location of the magnified areas.

Figure 2

Abundance changes with invasiveness, main mitochondrial proteins. (A) FAO enzymes. (B) ATP synthase subunits. (C) Cytochrome oxidase subunits. (D) TCA enzymes. Red bars represent increase and blue bars decrease, with light colors used for tendencies. Protein codes (for rattus norvegicus) are put in upper case and bold, and gene names in italics.

Figure 3

Distribution of ACADL expression in rat mesothelioma tumors. (A–D) Comparison of overall IHC staining with increasing invasiveness, ×400 (the scale bars represent 50 µm). (E) Magnifications of areas of intense staining in the most aggressive, M5-T1 tumor (the scale bars represent 25 µm).

Figure 4

Different expressions of mitochondrial genes between epithelioid and sarcomatoid MM cells. (A) Mitochondrial gene expression in 10 primary MM cell lines (Table 1) derived from two different histopathological subtypes, i.e., epithelioid (EPI, n = 5) and sarcomatoid (SAR, n = 5), was analyzed with real time PCR. Data are expressed as relative mean fold increase SAR vs. EPI MM cells. (B,C) MPM epithelioid (EPI UP1 and EPI UP2), and sarcomatoid (SAR UP6 and SAR UP7) cells were grown to confluence, then scratched and incubated for 24 h in fresh medium (CTRL) or medium with 10 µM of etomoxir (ETOM). (B) Representative bright-field images immediately after the scratch and after 24 h. (C) Cell migration. Data are presented as means ± SEM (n = 3). * p < 0.05, *** p < 0.001: ETOM treated cells vs. CTRL cells; # p < 0.05, ### p < 0.001: SAR cells vs. EPI cells. Scale bar is 100 µm.

Figure 5

Sarcomatoid MPM cells have higher expression of ACADL compared with epithelioid MM cells. Primary MM cells derived from two different histopathological subtypes, i.e., epithelioid (EPI UP1 and UP2) and sarcomatoid (SAR UP6 and UP7), were incubated in fresh medium (CTRL), or in medium with 10 µm of etomoxir (ETOM) for 24 h then used for measurements. (A) ACADL mRNA levels were measured with RT-PCR, in triplicate. Data are presented as means ± SEM (n = 3). ### p < 0.001: SAR cells vs. EPI cells. (B) ACADL protein was measured with immunoblotting in primary MM cell lines. GAPDH was used as a loading control. The figure is representative of one out of three experiments with similar results. The uncropped blots and molecular weight markers are shown in Supplementary Figure S1.

Figure 6

Sarcomatoid MM cells have more active mitochondrial metabolism compared with epithelioid MM cells. Primary MM cells derived from two different histopathological subtypes, i.e., epithelioid (EPI UP1 and UP2) and sarcomatoid (SAR UP6 and SAR UP7), were grown in fresh medium (CTRL) or in medium with 10 µM of etomoxir for 24 h and then used for the following analysis. (A) Fatty acid β-oxidation was measured with fluorimetric assay in triplicate. Data are presented as means ± SEM (n = 3). *** p < 0.001: ETOM treated cells vs. CTRL cells; # p < 0.05, ### p < 0.001: SAR cells vs. EPI cells. (B) The electron flux between Complex I and III was measured spectrophotometrically in triplicate. Data are expressed as means ± SEM (n = 3). * p < 0.05, *** p < 0.001: ETOM treated cells vs. CTRL cells, ### p < 0.001: SAR cells vs. EPI cells. (C) ATP release was measured with a chemiluminescence-based assay in duplicate. Data are expressed as means ± SEM (n = 3). * p < 0.05, *** p < 0.001: ETOM treated cells vs. CTRL cells; # p < 0.05, ### p < 0.001: SAR cells vs. EPI cells.