Sphingosine kinase 1 is required for TGF-β mediated fibroblast-to-myofibroblast differentiation in ovarian cancer

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.


Australian Ovarian Cancer Study (AOCS) and the Cancer Genome Atlas (TCGA) Data Analysis
Gene expression data from ovarian cancer patients in the AOCS [1] and TCGA [2] studies were analyzed using the R2 Genomic Analysis and Visualization Platform (http://r2.amc.nl). The molecular subtype specific expression of SPHK1 was analyzed in both datasets. The expression level of SPHK1 was correlated with the expression level of all the genes in each dataset. Genes with a Pearson correlation greater than or equal to 0.6 and a false discovery rate less than 5% were selected from each cohort (Supplementary Table S1). The list of correlated genes in each cohort was analyzed using R2 Gene Ontology Analysis at a false discovery rate of less than 5% (Figure 2A and Supplementary Table S2). For analysis of activated fibroblast marker expression (ACTA2, FAP) patients were stratified by median expression of SPHK1.

Isolation of fibroblasts and CAFs from human samples
The tumor and tissue specimen samples used in this study were obtained with informed consent from all subjects. Fibroblasts were isolated from primary human omentum obtained from patients with stage III/IV serous ovarian cancer. Normal omentum was defined as omentum that appeared grossly normal with no evidence of metastatic implants. Normal and cancerassociated fibroblasts were isolated using an adapted previously described protocol. [3] Briefly, fresh tissue was minced, suspended in a 1X solution of collagenase/ hyaluronidase (Stem Cell Technologies), and incubated for 6-8 hours on an orbital shaker at 37°C. After disassociation, the cell suspension was strained, and resulting suspension was centrifuged. The cell pellet was resuspended in DMEM with 10% FBS and 1% penicillin-streptomycin.

Coculture assays
For direct coculture, 2 X 10 6 fibroblasts were mixed with 1 X 10 6 GFP-labeled ovarian cancer cells (SKOV3ip1-luc GFP or OVCAR3-GFP) and cultured in a 10 cm dish for 48 hours. Cells were subsequently separated by FACS, and RNA was isolated. For indirect coculture, equal numbers of fibroblasts and ovarian cancer cells were cultured using a 0.4 μm Transwell insert.

Stimulation of cells with conditioned media
To generate tumor-conditioned media, 2 X 10 6 ovarian cancer cells were seeded in 10 cm dishes and cultured in serum-free DMEM medium for 48 hours. Tumor conditioned media was filtered using 0.45 μM syringe filter and stored at -80° until needed.

TGF-β1 and S1P quantitation by ELISA
Conditioned media obtained from ovarian cancer cells were subjected to the Human/Mouse TGF-β1 ELISA Ready-SET-Go assay following the manufacturer's protocol (eBioscience). S1P levels from fibroblast conditioned media were determined by the Sphingosine Kinase Activity Kit following the manufacturer's protocol (Echelon Biosciences).

Mouse Genotyping
Mouse genomic DNA was isolated from tail biopsies following overnight digestion at 55°C in 300 μL DirectPCR Lysis Reagent (Tail) (  with the SPHK1 inhibitor SKI-5C (5 μM) for 1 hour, and then stimulated with TGF-β1 for 48 hours. GAPDH was used as a loading control. The mRNA level of each gene is expressed relative to its level in vehicle treated cells. *p < 0.05 related to vehicle treated control; # p < 0.05 related toTGF-β1-stimulated expression values as indicated. B. Representative images of TGF-β-induced collagen gel contraction at 24 hours by TRS3 cells with and without pretreatment with SKI-5C. Data are presented as mean percent contraction compared to the total area of the well. C. TRS3 cells were incubated with S1PR1 inhibitor W146 (10 μM), S1PR2 inhibitor JTE-013 (10 μM), or S1PR3 inhibitor CAY10444 (10 μM) for 1 hour before TGF-β1 stimulation for 48 hours. qRT-PCR analysis of CAF-associated markers. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3 cells. *p < 0.05 related to vehicle treated control; # p < 0.05 related to TGF-β1-stimulated expression.
Supplementary Figure S10: The effect of TGF-β1 on the expression of S1P receptors (S1PRs) in ovarian fibroblasts. A.
Basal mRNA expression levels of all five S1P receptors in TRS3 and INOF cell lines were analyzed by qRT-PCR. Expression levels are normalized to RPL32. ND, not detected. B. mRNA expression levels of S1PR1, S1PR2, and S1PR3 upon TGF-β1 treatment for the indicated amounts of time. * p-value < .01.