Sphingosine 1-phosphate signalling contributes to transforming growth factor-β1-induced myofibroblast differentiation in oral cancer.

The prognosis for patients with oral squamous cell carcinoma (OSCC) is poor and novel ways to manage the disease are required. The density of myofibroblastic cancer-associated fibroblasts (myoCAFs) within the OSCC stroma correlates with poor prognosis, suggesting that myoCAFs could be targetted therapeutically. OSCC cells have been shown to regulate myoCAF differentiation via mechanisms involving transforming growth factor-β1 (TGF-β1), but the further downstream mechanisms are yet to be clearly defined. In this study, we aimed to examine the involvement of sphingosine 1-phosphate (S1P) signalling in mediating myofibroblast differentiation. Treatment of normal oral fibroblasts with OSCC conditioned media (CM) or TGF-β1 induced the expression of α-smooth muscle actin (α-SMA), a well-recognised marker of fibroblast activation/myofibroblasts. TGF-β1 up-regulated expression of sphingosine kinase 1 (SK1), but not sphingosine kinase 2 (SK2). However, inhibiting either of the SKs abrogated the induction of α-SMA expression by TGF-β1 and OSCC CM. The effect of inhibiting SK1/2 could not be rescued by exogenous S1P suggesting a possible role for intracellular S1P. Normal oral fibroblasts were shown to express predominantly sphingosine 1-phosphate receptor 2 (S1PR2) and sphingosine 1-phosphate receptor 3 (S1PR3) receptors. The induction of α-SMA expression by OSCC CM was abrogated with a S1PR2 antagonist, whilst α-SMA expression was upregulated following treatment with a S1PR2 agonist. Using pharmacological modulation of α-SMA expression, our data show that SKs and S1PR2 contribute to myofibroblast differentiation in OSCC. These results highlight the potential of inhibiting S1P signalling to inhibit myoCAF development.