Phospho-S1P1 T236 Levels, but not Total S1P1, are Elevated in Human TNBC Cells. (a–c) Relative transcript levels of SPHK1 (a), SPHK2 (b), and S1P1 (c) in human TNBC (n = 41) and non-TNBC samples (n = 385) from a reanalyzed microarray dataset (GSE2034; [24]). (d) Western blot analysis of phospho-S1P1 T236 (P-S1P1 T236), total S1P1, and ACTIN levels in human TNBC (n = 6) and luminal (n = 10) cell lines with MCF10A nontransformed cell line as a control. (e–g) S1P1 (e) and P-S1P1 T236 (f) to ACTIN as well as P-S1P1 T236 to S1P1 ratio (g) in TNBC and luminal breast cancer cell lines. Data indicate mean ± SEM.

Increased Levels of P-S1P1 T236 are linked with TNBC Progression. (a) Western blot analysis of SPHK1, P-S1P1 T236, S1P1, phospho-AKT S473 (P-AKT S473), AKT, and ACTIN levels in human MI, NeoT, MIII, and MIV cells. (b) Overlay of brightfield and fluorescence images of zebrafish embryos transplanted with RFP⁺ human MI, NeoT, MIII, and MIV cells at 3 days post-transplantation (dpt; n = 3 per group). The scale bar represents 200 µm. (c) Quantification of invasive spread in zebrafish embryos based on fluorescence intensity of MI, NeoT, MIII, and MIV cells (n = 3 per group). Data presented as Mean ± SEM. NS, not significant.

P-S1P1 T236 contributes to the distant spread of TNBC. (a) Western blotting analysis of P-S1P1 T236, S1P1, and ACTIN in MIII and MIV cells overexpressing Luciferase, S1P1 WT, or the phosphorylation-defective S1P1 T236A mutant. (b) Overlay of brightfield and red fluorescence images of zebrafish xenografts transplanted with RFP⁺ MIII and MIV cells overexpressing Luciferase, S1P1 WT, or S1P1 T236A at 3 dpt (right panel). (c) Quantification of invasive properties of MIII and MIV cells overexpressing Luciferase, S1P1 WT, or S1P1 T236A in zebrafish xenografts based on fluorescence intensity of RFP⁺ tumor cells outside of the initial injection area (n = 6 for MIII and MIV overexpressing Luciferase, and n = 3 for MIII and MIV overexpressing S1P1 WT or S1P1 T236A). (d,e) Wound-scratch assay (d) and quantification (e) show increased migration properties of MIII cells overexpressing S1P1 WT though not S1P1 T236A at 21 h after wound scratch, compared to control Luciferase-overexpressing cells (n = 3 per group). Data are presented as Mean ± SEM. Scale bar = 200 µm in (b) and Scale bar = 5 μM in (d).

AKT Inhibitor MK2206 reduces P-S1P1 T236 and impairs TNBC invasion in Zebrafish xenografts. (a) Western blot analysis of P-S1P1 T236, S1P1, P-AKT S473, AKT, and ACTIN in MIII and MIV cells without and with MK2206 (90 nM) treatment. (b) Quantification of fluorescence intensity of tumor burden in (c, n = 3 per group). (c) Overlay of brightfield and fluorescence images of zebrafish embryos transplanted with RFP⁺ MIII and MIV cells and treated with DMSO or MK2206 (0.3 μM) at 3 dpt. Scale bar = 200 µm. Data are presented as Mean ± SEM.

AKT-mediated phosphorylation of S1P1 T236 drives TNBC invasion. (a) Western blot analysis of P-AKT S473, AKT, P-S1P1 T236, and S1P1 expression in MIII cells overexpressing Luciferase, S1P1 WT, or S1P1 T236A in the presence or absence of 0.3 μM MK2206 treatment. P-AKT and AKT to ACTIN ratio are shown as relative values, with the vehicle-treated cells overexpressing Luciferase set as 100. (b,c) Wound-scratch assay (b) and data quantification (c) Reveal migration characteristics of MIII cells overexpressing Luciferase, S1P1 WT, or S1P1 T236A treated with DMSO or MK2206 (90 nM) at 0 and 19 h post scratching, showing that MK2206′s ability to inhibit migration of TNBC cells depends on AKT-mediated phosphorylation of S1P1 T236 (n = 3 per group). (d) Overlay of brightfield and red fluorescence images of zebrafish xenografts transplanted with RFP⁺ MIII cells overexpressing Luciferase or S1P1 WT treated with DMSO or MK2206 (0.3 μM) at 3 dpt. (e) Quantification of fluorescence intensity of invasive tumor cells outside the injection area (n = 3 per group). Scale bar = 200 µm in (b) and Scale bar = 5 µM in (d). Data are presented as Mean ± SEM.

FTY720 induces apoptosis and suppresses the distant spread of human TNBC cells with high P-S1P1 T236 Levels. (a) Western blot analysis of P-S1P1 T236, S1P1, P-AKT S473, AKT, active CASPASE 3 (CASP3), and ACTIN levels in MI, NeoT, MIII, and MIV cells before and after FTY720 treatment (2 μM). (b) Brightfield images of MI, NeoT, MIII, and MIV cells at 48 h post-treatment with DMSO or FTY720 (n = 3 per group). The scale bar represents 1 µM. (c) The proportion of viable cells in response to a dose gradient of FTY720 shows a significantly reduced viability of MIII and MIV though not MI nor NeoT cells (n = 3 per group). (d) Overlay of brightfield and fluorescence images of zebrafish embryos injected with RFP⁺ MIII and MIV cells treated with DMSO or FTY720 (2 μM) at 3 dpt (n = 3 per group). The scale bar represents 200 µm. (e) Quantification of fluorescent intensity of tumor cells described in (d) Shows FTY720′s ability to inhibit the distant spread of TNBC cells (n = 3 per group). Data indicate mean ± SEM.