The influence of sphingosine-1-phosphate receptor 2 in determining the invasive behaviour of oral squamous carcinoma cell lines using 2 and 3D models

Wongviriya, Adjabhak (2023). The influence of sphingosine-1-phosphate receptor 2 in determining the invasive behaviour of oral squamous carcinoma cell lines using 2 and 3D models. University of Birmingham. Ph.D.

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Abstract

Introduction: The sphingosine-1-phosphate receptor 2 (S1PR2) has been shown to influence several cellular activities, including growth and motility. Epidermal growth factor (EGF) is well-known for its ability to regulate the proliferation and motility of various cell types. In previous studies, the activation of S1PR2 suppressed tumour propagation in various cell types; however in oral squamous cell carcinoma (OSCC) lines, it stimulated migration and invasion. Since a single study may not be enough to clarify the function of S1PR2 and the overlapping functions between S1PR2 and EGF, the present study investigated the influence of EGF and S1PR2 in proliferation, migration and invasion in three OSCC lines (H357, H400 and H413) as well as potential crosstalk between EGF/ EGF receptors and S1P/S1PR2 pathways.

Material and methods: Proliferation in response to EGF and S1PR2 treatments was determined using growth curves and the BrdU assay. Migration was studied using scratch wound and transwell assays, whilst matrix invasion was investigated using a transwell assay and a multicellular tumour spheroid (MCTS) model. EGF production was determined using an enzyme-linked immunosorbent assay (ELISA) and gene expression levels were determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analyses.

Results: While cultures treated with 1 ng/ml EGF promoted the proliferation of H357 and H413 and inhibited H400 cells, 20 ng/ml EGF inhibited the growth of all three OSCC lines. Inhibition of S1PR2 with the known antagonist JTE013 decreased proliferation but stimulation with the agonist CYM5478 did not increase proliferation. EGF at 1 and 20 ng/ml induced migration and invasion of all three OSCC lines with the concentrations applied. S1PR2 inhibition reduced migration and invasion of these lines. The reverse effect on migration and invasion was identified when S1PR2 was stimulated except for H357 and H413 in scratch wound assays and for H400 in transwell assays. S1PR2 also suppressed EGF’s induction of migration and invasion. Neither the inhibition nor stimulation of SIPR2 affected EGF production, however EGF stimulation was found to upregulate sphingosine kinase 1 (SPHK1), an enzyme that generates S1P.

Conclusions: The three OSCC lines exhibited different characteristics of proliferation, migration, invasion and gene expression. EGF regulated OSCC proliferation to differing degrees, depending on the concentration and cell line, but it promoted migration and invasion according to concentration. The inhibition of S1PR2 suppressed proliferation, migration and invasion of the three OSCC lines and also the EGF-induced migration and invasion effect. This implied that S1P/S1PR2 may exert control on EGF/ EGF receptors in regulating the motility of the three OSCC lines. EGF/EGF receptors also demonstrated crosstalk with the S1P/S1PR2 axis by increasing the expression of SPHK1, which potentially increases S1P production. This information about the role of S1PR2 in regulating growth, migration and invasion and how it involves EGF/EGF receptors may lead to promising new treatments for OSCC patients by potentially preventing or reducing metastasis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):

Supervisor(s)	Email	ORCID
Landini, Gabriel	UNSPECIFIED	UNSPECIFIED
Shelton, Richard M.	UNSPECIFIED	UNSPECIFIED
Cooper, Paul	UNSPECIFIED	UNSPECIFIED
Milward, Michael	UNSPECIFIED	UNSPECIFIED