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

Abstract

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.

Keywords: cancer-associated fibroblast (CAF); microenvironment; sphingosine kinase 1 (SPHK1); sphingosine-1-phosphate (S1P); transforming growth factor-beta 1 (TGF-β1).

Figure 1. High SPHK1 expression is associated with reduced survival of patients with HGSC

A. Quantification of SPHK1 mRNA in benign ovaries (n = 7) and ovarian cancer (n = 77) patient samples by OpenArray Real-Time PCR. Expression levels were normalized to RPLP0. Statistical significance was determined by Mann Whitney U test.*p < 0.05. B. Kaplan-Meier plot analysis of progression-free and overall survival of patients stratified by SPHK1 transcript levels (Affymetrix ID: 219257_s_at) in a combined cohort of 13 gene expression datasets. Low and high SPHK1 expression were defined by the auto-calculated best cutoff. Significance values were determined by log-rank test. HR indicates the hazard ratio, and “Low” and“High” in parentheses indicate the number of cases per group.

Figure 2. SPHK1 expression is associated with reactive stroma in ovarian cancer

A. GO enrichment analysis of genes that correlate with SPHK1 expression (Pearson correlation, R ≥ 0.6) in the AOCS dataset (n = 285). B. SPHK1 transcript levels associated with the classified molecular subtypes of ovarian cancer by Tothill et al. in the AOCS dataset. C. Frequency of C1 and C4 molecular subtypes among SPHK1-Low and SPHK1-High cases in the AOCS dataset. The C1 molecular subtype is characterized by a reactive stromal signature, while the C4 molecular subtype is described as having a low stromal response signature. Median expression was used to define SPHK1-Low (n = 143) and SPHK1-High (n = 142) samples. D. Box-and-whisker plots of the differences in transcript levels of ACTA2 (encoding αSMA) and FAP, between SPHK1-Low and SPHK1-High tumors in the AOCS dataset. Statistical significance was determined by Mann Whitney U test. E. Plot showing the expression level of SPHK1 in laser capture-microdissected stromal fibroblastic and epithelial components of both normal and malignant ovarian tissue samples (GSE40595). Statistical significance was determined by Mann Whitney U test. In all box-and-whisker plots, horizontal bars indicate the medians, boxes indicate the 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. *p < 0.05. CAFs, cancer-associated fibroblasts; OSE, ovarian surface epithelium.

Figure 3. Ovarian cancer cells stimulate SPHK1 expression in ovarian fibroblasts via TGF-β1 signaling

A, B. qRT-PCR analysis of SPHK1 mRNA expression in TRS3 ovarian fibroblasts cells A. cultured alone or cocultured with fluorescently labeled ovarian cancer cells for 48 hours, followed by FACS or B. stimulated with non-conditioned media (control) or conditioned media from ovarian cancer cells for 48 hours. C. Transcript levels of CAF markers in TRS3 cells incubated in ovarian cancer cell conditioned media for 48 hours. The mRNA level of each gene is indicated relative to its level in TRS3 cells incubated in non-conditioned media. D. Transcript levels of CAF markers in TRS3 cells incubated in ovarian cancer cell conditioned media for 6 hours with either anti-TGF-β antibody (α TGF-β) or IgG control. E. Induction of CAF markers by ovarian cancer conditioned media in TRS3 cells with or without pretreatment with the TGF-β type I receptor inhibitor A83-01. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3 cells (control). F. TRS3 cells were treated with TGF-β1 for the indicated times and mRNA (upper panel) and protein (lower panel) were harvested and evaluated for SPHK1 expression. G. Transcript levels of CAF-associated genes in TRS3 cells stimulated with TGF-β1 for 48 hours with or without pretreatment with A83-01. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3 cells. Data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05. CM, conditioned media.

Figure 4. Overexpression of SPHK1 in ovarian fibroblasts enhances TGF-β1-induced myofibroblast marker expression and their ability to promote tumor cell migration and invasion

A. Western blot and B. qRT-PCR analysis of CAF markers in TRS3-GFP and TRS3-SPHK1 cells 48 hours after stimulation with TGF-β1. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3-GFP cells. *p < 0.05 related to vehicle treated control; ^#p < 0.05 related to TGF-β1-stimulated expression values as indicated. C. Immunofluorescence staining of αSMA in TRS3-GFP and TRS3-SPHK1 cells with and without TGF-β1 stimulation. Scale bar, 100 μm. D. Transwell migration of TRS3-GFP and TRS3-SPHK1 cells. Data are shown as fold change normalized to TRS3-GFP cells. E. Representative images of collagen gel contraction by TRS3-GFP and TRS3-SPHK1 cells at 24 hours. F. Quantification of collagen gel contraction. Data are presented as mean percent contraction compared to the total area of the well. G. SKOV3 or OVCAR3 cells were cocultured with TRS3-GFP or TRS3-SPHK1 cells for 48 hours. Migration and invasion of isolated ovarian cancer cells was assessed by transwell and matrigel invasion assay, respectively. Data are expressed as fold change normalized to ovarian cancer cells cocultured with TRS3-GFP cells. All data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05. NS, not significant.

Figure 5. Knockout of SPHK1 in ovarian fibroblasts inhibits TGF-β1-induced myofibroblast differentiation and CAF-like function

A. Western blot and B. qRT-PCR analysis of CAF markers in TRS3-sgCONT and TRS3-sgSPHK1 cells 48 hours after stimulation with TGF-β1. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3-sgCONT cells. *p < 0.05 related to vehicle treated control; ^#p < 0.05 related to TGF-β1-stimulated expression values as indicated. C. Immunofluorescence staining of αSMA in TRS3-sgCONT and TRS3-sgSPHK1 cells with and without TGF-β1 stimulation. Scale bar, 100 μm. D. Transwell migration of TRS3-sgCONT and TRS3-sgSPHK1 cells. Data are shown as fold change normalized to TRS3-sgCONT cells. E. Representative images of collagen gel contraction by TRS3-sgCONT and TRS3-sgSPHK1 cells at 24 hours. F. Quantification of collagen gel contraction. Data are presented as mean percent contraction compared to the total area of the well. G. SKOV3 or OVCAR3 cells were cocultured with TRS3-sgCONT or TRS3-sgSPHK1 cells for 48 hours. Migration and invasion of isolated ovarian cancer cells was assessed by transwell and matrigel invasion assay, respectively. Data are expressed as fold change normalized to ovarian cancer cells cocultured with TRS3-sgCONT cells. All data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05. NS, not significant.

Figure 6. Pharmacological inhibition of SPHK1 or S1PR2/3 signaling inhibits TGF-β1-induced myofibroblast differentiation

A. Western blot and B. qRT-PCR analysis of CAF markers in TRS3 cells pretreated with the SPHK1 inhibitor SKI-5C (5 μM) for 1 hour, and then stimulated with TGF-β1 for 48 hours. 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 to TGF-β1-stimulated expression values as indicated. C. 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. D. TRS3 cells were incubated with S1PR1 antagonist W146 (10 μM), S1PR2 antagonist JTE-013 (10 μM), or S1PR3 antagonist CAY10444 (10 μM) for 1 hour before TGF-β1 stimulation for 48 hours. qRT-PCR analysis of CAF markers where 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 to TGF-β1-stimulated expression.

Figure 7. TGF-β1 induces p38 MAPK phosphorylation through S1PR2 and S1PR3 in ovarian fibroblasts

A. Western blot analysis of phospho-p38 in TRS3-sgCONT and TRS3-sgSPHK1 after TGF-β1 stimulation for the indicated times. B. Western blot analysis of phospho-p38 in TRS3 cells pretreated with either S1PR1 antagonist W146 (10 μM), S1PR2 antagonist JTE-013 (10 μM), or S1PR3 antagonist CAY10444 (10 μM) for 1 hour, and then stimulated with TGF-β1 for 30 minutes. C. qRT-PCR analysis of CAF markers and ECM-associated genes in TRS3 cells pretreated with the p38 MAPK inhibitor SB203580 (10 μM) for 1 hour, and then stimulated with TGF-β1 for 48 hours. D. Western blot analysis of phospho-p38 in TRS3-sgSPHK1 cells pretreated with S1P (100 nM) for 1 hour before the addition of TGF-β1 for 30 minutes. E. qRT-PCR analysis of CAF markers in TRS3-sgSPHK1 cells pretreated with S1P (100 nM) for 1 hour, and then stimulated with TGF-β1 for 48 hours. For western blots, relative band intensities of phosphorylated proteins were quantified by densitometry, and the ratios of the phosphorylated to total signals are indicated below the blot. For each cell line the ratios are expressed as fold change from the untreated control. For qRT-PCR, 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 to TGF-β1-stimulated expression.

Figure 8. Stromal SPHK1 expression modulates tumor growth and dissemination in a mouse model of ovarian cancer

A. Comparison of survival rates of female Sphk1^−/− and wild type (Sphk1^+/+) mice after intraperitoneal injection of 5×10⁶ HRas-MOSE cells per mouse (n = 9 mice per group). B. Table summarizing the median survival time and number of mice per group that developed tumors, carcinomatosis, and diaphragm metastases. C. Representative photographs of tumor burden in female Sphk1^−/− and wild type mice. White arrowheads indicate tumor nodules.