Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis

Background: Colorectal cancer (CRC) progression and related mortality are highly associated with metabolic disorders. However, the molecular mechanism involved in the regulation of hyperlipidemia-associated CRC metastasis remains unclear. This study aimed to investigate the effects of angiopoietin-like 4 (ANGPTL4) on NADPH oxidase 4 (NOX4) expression and reactive oxygen species (ROS) production, which might provide new targets for improving outcomes in patients with hyperlipidemia-associated CRC metastasis. Methods: The clinical relevance of relationship between NOX4 expression and ANGPTL4 was examined in CRC patients by the Oncomine and TCGA data set. Expressions of NOX4, epithelial-mesenchymal transition (EMT) markers, and gene regulation of NOX4 in free fatty acids (FFAs)-treated CRC cells were determined. The FFAs-triggered metastatic ability of CRC cells under treatments of antioxidants or knockdown of NOX4, ANGPTL4, and MMPs was evaluated in vitro and in vivo. In addition, effects of antioxidants and depletion of metastasis-associated molecules on the correlation between ROS production and FFAs-promoted CRC metastasis were also clarified. Results: In this study, we found that the induction of NOX4, followed by the increased ROS was essential for oleic acid (OA)-promoted CRC cell metastasis. The depletion of ANGPTL4 significantly inhibited c-Jun-mediated transactivation of NOX4 expression, accompanied with reduced levels of ROS, MMP-1, and MMP-9, resulting in the disruption of OA-promoted CRC cell metastasis. Moreover, knockdown of ANGPTL4, NOX4, MMP-1, and MMP-9 or the treatment of antioxidants dramatically inhibited circulating OA-enhanced tumor cell extravasation and metastatic seeding of tumor cells in lungs, indicating that the ANGPTL4/NOX4 axis was critical for dyslipidemia-associated tumor metastasis. Conclusion: The coincident expression of NOX4 and ANGPTL4 in CRC tumor specimens provides the insight into the potential therapeutic targets for the treatment of dyslipidemia-associated CRC metastasis.

Briefly, 1 × 10 5 cells were seeded to each well in 6-well plates for overnight. Before infection of lentivirus, the culture media was changed to the fresh media containing 8 μg/ml polybrene (Sigma-Aldrich). Lentivirus was added to the cells at MOI = 3.

Real-time quantitative PCR
Following cDNA synthesis, gene-specific primers were designed using NCBI primer design software. Real-time quantitative PCR was performed in triplicate using a SYBER Green MasterMix (Invitrogen) and an StepOne Real-Time PCR Systems

Western blotting
An analytical 12% SDS-PAGE was performed, and 30 μg of protein of each were analyzed. Western blotting was performed as previously described [1]. Antibodies against human NOX4 (GTX121929) and MMP-9 (GTX100458) (GeneTex, Hsinchu, were used as the primary antibodies.

CFSE proliferation assay
Cells were harvested and resuspended in 1 ml medium. Cells were labelled by the addition of 1 ml of PBS containing 5 μM the carboxyfluorescein succinimidyl ester (CFSE), followed by 5 min incubation at room temperature. Labelled cells were then washed twice, counted and seeded at 1 × 10 5 cells/well in a 12 well plate. On the time of analysis for 24, 48, 72 h, cells were harvested and washed twice with PBS and then CFSE intensity was examined by FACS. Experiments were performed at least three times and data presented are of one representative experiment.

Quantitative estimation of H 2 O 2 concentration
The quantitative estimation of H2O2 was carried out using a Hydrogen Peroxide Assay kit (Cat. No. K265-200) purchased from BioVision (BioCat, Heidelberg, Germany).
Cells was lysis in 0.5 mL of PBS containing 1% Triton X-100. Cell lysates was then at 10,000 g at 4°C for 30 min and supernatant was used immediately for quantitative estimation of H2O2. The optical density (OD) was determined using a microplate reader set to 550 nm. Samples from three independent experiments were used for each group and all samples were run in one assay to avoid inter-assay variation.

RNA stability assay
To determine the half-life of OA-induced mRNA expression of gene, cells were treated with OA for 3 h, and then the 5 μg/ml Actinomycin D (Sigma) was added into the growth medium to block transcription. During the following 3, 6 ,9 h, cells were harvested and total RNA was prepared by TRIzol® (Invitrogen Life Technologies). cDNA was prepared by reverse transcriptase reaction by using reverse transcriptase reaction kit (Invitrogen Life Technologies) according to the manufacturer's instructions and the expression of target genes were analyzed by real-time quantitative PCR.

DNA affinity precipitation assay
Quantitation of the change of c-Jun binding to the NOX4 promoter element was achieved by a DNA affinity precipitation assay (DAPA) according to the method as previously described [2]. In brief, 5'-biotinylated oligonucleotides and corresponding to the sense -4679 to -4650 bp and antisense strands of the NOX4 promoter element

Plasmid construction
Truncated lengths of NOX4 promoter were constructed by primer sets in the previous study [3]. Especially, NOX4 promoter (1.84 kb) was amplified with primer sets: sense, (DN-IκB) mutant was generated by N-terminal deletion of residues 1-45 using a standard PCR approach [5].