Study of transcriptional effects of FOXS1 silencing and TGF-beta treatment in SNU-449 hepatocellular carcinoma cell line

Background & aims: Transforming Growth Factor beta (TGFβ) is a pleiotropic cytokine which controls fundamental cellular processes associated with tumor onset and progression. In hepatocellular carcinoma (HCC), we previously highlighted TGFβ signatures recapitulating its functional duality (i.e. cytostatic versus pro-metastatic features) to discriminate patients with good and poor prognosis. Here, we aimed at characterizing Forkhead Box S1 (FOXS1), a predicted transcription factor that we hypothesize to mediate pro-metastatic properties of TGFβ. Methods: FOXS1 expression was profiled in well-characterized HCC cell lines. Transient loss of function of FOXS1 (siRNA) was combined with functional assays and analysis of epithelial-mesenchymal transition (EMT) markers by western blot and immunofluorescence. Clinical relevance of FOXS1 was determined by integrative genomics. Results: Our data show that FOXS1 is a novel canonical SMAD-dependent TGFβ target. Its expression correlates with a mesenchymal phenotype. FOXS1 mediates pro-metastatic properties of TGFβ by inducing EMT and cell migration. In human HCC, FOXS1 correlates with VIM expression and is highly expressed in specific molecular subtypes associated with a poorer differentiation and no CTNNB1 mutation. FOXS1 expression predicts a poor prognosis (e.g. reduced patient survival) in liver, colon, stomach and kidney cancer. Conclusions: FOXS1 is a novel TGFβ target and a key mediator of EMT. Its induction in cancer is associated with a poor prognosis. Thus, determining the expression of FOXS1 in advanced tumors in which the pro-metastatic arm of TGFβ is activated could help to identify patients who may benefit from targeted therapies using TGFβ inhibitors. SNU-449 cells (ATCC® CRL-2234, grade II-III/IV) were grown in a MEM medium (Sigma, M2279) supplemented with 100U/ml penicillin, 100μg/ml streptomycin (Gibco, 15070-063), 2mM L-Gluta and 10% fetal bovine serum (HyClone). Cultures were performed at 37°C in a 5% CO2 atmosphere. Overnight serum starvation was performed before each treatment. Cells were treated with 1ng/mL recombinant human TGFβ1 (R&D system, Minneapolis, USA) versus BSA control for 72hrs. FOXS1 loss of function was performed by using two different small interfering RNAs (siRNA) purchased from Ambion: siFOXS1#1 (s5256, 5’-AAUCGGCCCAUGAUGUAGCgg-3’) and siFOXS1#2 (s107325, 5’-CAUGCAAAAAUUCAGUGCCgg-3’). FAM-labeled negative control siRNA No.1 (Ambion AM4620) was used as control (siNC). Cells were transfected at 70% confluency with 50nM siRNA using lipofectamine RNAiMAX reagent and Opti-MEM medium according to manufacturer’s instructions (Invitrogen). Experiments were performed in triplicates.