miR-497-5p mediates starvation-induced death in colon cancer cells by targeting acyl-CoA synthetase-5 and modulation of lipid metabolism.

Modulation of fatty acids metabolism is an appropriate strategy for starvation-induced death in tumor cancers. Colon cancer cells express a high level of acyl-CoA synthetase-5 (ACSL5), and as yet no therapeutic approach has been achieved. Herein, ACSL5-related microRNAs (miRNAs) were identified via TargetScan, and their impacts on ACSL5 and lipid content along with metabolic activity, cell cycle, migration, and invasion of colorectal cancer (CRC) cells were examined, and subsequently compared with transcriptome for better visualization of intracellular-signaling networks. In vivo analysis was performed using BALB/c mice xenograft model of CRC injected with target miRNA. Clinical significances were also evaluated in 80 CRC tumors and matched adjacent normal tissues. There was a reverse correlation between ACSL5 and miR-497-5p, which miR-497-5p overexpression modulated CRC cell proliferation and development. A similar observation was received from the in vivo examination in which intratumoral injection of miR-497-5p reversed the tumor growth in the CRC xenograft model. Downregulation of miR-497-5p correlated with tumor differentiation, tumor, node, and metastasis staging, lymph node metastasis, and poor survival in patients with CRC. These results suggested that miR-497-5p upregulation could be considered as a therapeutic strategy for modulation of lipid metabolism in colon cancer.

Low acyl-CoA synthetase 5 expression in colorectal carcinomas is prognostic for early tumour recurrence.

It has been shown that the metabolism of long chain fatty acids is involved in colorectal carcinogenesis. Acyl-CoA synthetases (ACSL) activate free fatty acids by synthesis of acyl-CoA thioesters. ACSL isoform 5 (ACSL5) is involved in enterocytic differentiation and maturation by regulating both pro-apoptotic and anti-proliferative effects. Whilst impaired expression of ACSL5 has been associated with sporadic colorectal carcinogenesis, little is known about ACSL5 as a prognostic factor. Aim of this retrospective study was to characterize the prognostic impact of ACSL5 expression levels in sporadic colorectal adenocarcinomas. A total of 72 patients with a median follow-up of 54 months was included. Using a standardized immunohistochemical approach, colorectal adenocarcinomas with low (n=41; group 1) or high (n=31; group 2) ACSL5 levels were identified. In a one-year follow-up, tumour recurrence was significantly increased in group 1 (p=0.0279). The finding was independent of the TNM- and UICC-stage in the surgical resections. Frequency of lymph node metastasis and mortality was not different between the groups. In a long-time follow-up no differences were found between the ACSL5 groups. The data indicate that ACSL5 could be an independent prognostic factor for early recurrence of sporadic colorectal adenocarcinoma.

Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C.

AIM: To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C. METHODS: The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 µL reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysates. Reactions were terminated after 10, 30 or 60 min of incubation with Doles medium. RESULTS: Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl beta-D-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni(2+)-affinity chromatography. Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5, as well as recombinant rat ACSL1 (sensitive control), but not recombinant rat ACSL5 (insensitive control). The IC50 for human ACSL5 was about 10 µmol/L. The inhibitory triacsin C effect was similar for different incubation times (10, 30 and 60 min) and was not modified by the N- or C-terminal location of the 6xHis-tag. In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment, stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed. In both models, ACSL5 peak activity was found at pH 7.5 and pH 9.5, corresponding to the properties of recombinant human ACSL5 protein. In the presence of triacsin C (25 µmol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa. CONCLUSION: The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.